KR20210025063A - Anti-Marinobufagenin Antibodies and Uses thereof - Google Patents

Abstract

The present disclosure provides chimeric, humanized and human monoclonal antibodies (mAb) and fragments thereof that specifically bind marinobufagenin (MBG) with high affinity. Anti-MBG mAbs and fragments thereof can be used to treat MBG-related disorders, including hypertensive disorders and fibrotic disorders, optionally in combination with additional therapeutic agents. In addition, anti-MBG mAb and fragments thereof can be used for diagnostic purposes, including detecting MBG in biological samples and diagnosing MBG-related diseases.

Description

Anti-Marinobufagenin Antibodies and Uses thereof

(Statement on federally supported research)

The present invention is granted by the government agency NIH/NICHD (National Institute of Child Health and Development), Grant No. It was made with government support according to 1R43HD072745-01. The government has certain rights in the invention.

(Sequence list, table, or reference to a computer program)

The official copy of the sequence listing will be submitted at the same time as the specification as a text file in ASCII format via EFS-Web, the file name is "PRI002_ST25.txt", the creation date is June 7, 2018, and the size is 52 kilobytes. Sequence listings submitted through EFS-Web are part of the specification and the entirety is incorporated herein by reference.

Hypertension is associated with a wide range of disorders. One of the disorders of hypertension is pre-eclampsia (PE). Preeclampsia (PE) is a serious pregnancy complication characterized by hypertension (diastolic 90 mmHg or more) and proteinuria (300 mg or more at 24 hours) after 20 weeks of pregnancy. Pre-eclampsia (PE) is sometimes or often caused by swelling (e.g., pulmonary edema), encephalopathy, seizures, renal insufficiency, liver failure, intrauterine growth restriction (IUGR). ) And premature birth, the latter two are detrimental to the survival and well-being of the fetus and newborn. Pre-eclampsia (PE) occurs in about 5-10% of pregnancies and is a leading cause of maternal and fetal morbidity and mortality worldwide. The only known effective treatment for pre-eclampsia (PE) is early delivery of newborns.

In addition, fibrosis contributes to many diseases. One of the fibrotic diseases is diabetic nephropathy (DN) characterized by high blood pressure. Diabetic kidney disease (DN) affects about 15-25% of people with type 1 diabetes and about 30-40% of people with type 2 diabetes, and is the most common end-stage renal disease (ESRD) in the world. ). Clinical diabetic kidney disease (DN) is characterized by an initial increase in intraglomerular capillary pressure and glomerular filtration rate (GFR), glomerular hypertrophy, mesangial matrix expansion, and glomerular. Thickening of the glomerular basement membrane, arteriolar hyalinosis, nodular glomerulosclerosis, tubulointerstitial fibrosis and damage, and proteinuria (including albuminuria). It entails a series of pathological events. For example, scarring of glomerular capillaries (glomerular sclerosis) impairs the filtration process of the renal corpuscle and causes proteinuria by leaking proteins from the blood into the urine. Poor glycemic control in patients with diabetic kidney disease (DN) leads to a decrease in GFR, which can lead to ESRD, and worsens disease progression to kidney failure.

The present disclosure provides antibodies comprising fragments thereof that specifically bind marinobufagenin (MBG) with high affinity. These antibodies can be monoclonal and can be human, chimeric or humanized antibodies. Chimeric anti-MBG antibodies comprising fragments thereof are non-human (e.g., murine) complementarity-determining region (CDR) and non-human framework region(s), and optionally one or more human constants. It can have a human constant domain. Humanized anti-MBG antibodies comprising fragments thereof include non-human (e.g., murine) CDRs and human framework region(s), and optionally non-human framework amino acid residues adjacent to the CDRs and optionally one or more human It can have a constant domain. Human anti-MBG antibodies and fragments thereof may have human CDRs and human framework region(s), and optionally one or more human constant domains.

The disclosed human antibody represents a naive of antibodies that bind to marinobufagenin, an anti-marinobufagenin antibody from a library representing the human genome repertoire. Panning in the human phagemid library (naive of antibodies, representing the human genome repertoire) identified dozens of MBG-specific clones. Subsequent characterization of more than 100 human antibodies (Fabs) reduced human antibodies to 14 native antibodies that appeared to bind MBG in a competitive ELISA screen. Therefore, the human antibody obtained from the library represents an antibody found in a naive, human genomic library capable of binding to marinobufagenin.

Anti-MBG antibodies described herein may include modifications that provide the desired properties of the antibody. For example, the modification can increase the serum half-life of the antibody, or the modification can decrease the serum half-life. In addition, the modification can increase the effector function of the antibody, for example, increase the elimination of the antibody-MBG complex. Modification can reduce immunogenicity, or can reduce other undesired side effects or deleterious effects caused by anti-MBG antibodies.

Anti-MBG antibodies described herein can negate or inhibit the biological actions and effects of MBG, and thus can be used to treat MBG-related diseases. For example, anti-MBG antibodies described herein can be used to inhibit receptor binding by MBG on cells. The antibodies described herein can be used to inhibit the nuclear translocation of PKC-δ induced in cells by marinobufagenin. Antibodies described herein can be used to reduce the inhibition of proliferation by endothelial cells caused by marinobufagenin. The antibodies described herein can be used to reduce the inhibition of angiogenesis by endothelial cells caused by marinobufagenin. MBG-related diseases can include, for example, acute respiratory distress syndrome, and/or traumatic brain injury. Other MBG-related diseases include, for example, hypertension (e.g., essential hypertension), hypertension-related diseases (e.g., pre-eclampsia), and fibrosis-related diseases (e.g., non Alcoholic steatohepatitis, diabetic nephropathy). Such MBG-related diseases can be treated by administering to a subject suffering from an MBG-related disease one or more of the antibodies described herein.

Anti-MBG antibodies described herein can be used for diagnostic purposes. This MBG diagnosis uses antibodies to detect MBG. This diagnosis can be performed in vitro or in vivo. Such a diagnosis can, for example, quantify MBG in a sample or detect the location of MBG and/or the concentration of MBG at that location. This diagnosis can be used to determine whether a subject is suffering from an MBG-related disease disorder, and, optionally, the severity of an MBG-related disease.

A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which presents exemplary aspects of the present disclosure, and the accompanying drawings.
1 shows antigen binding of humanized anti-MBG in direct ELISA binding experiments. FIG. 1A compares six humanized anti-MBG antibodies, while FIG. 1B compares humanized anti-MBG and parent 3E9 antibody.
Figure 2a is a protein kinase C-delta (PKC-δ, 78 kDa) in the nucleus of primary cardiac fibroblasts cultured with MBG alone (2 nM) or with MGB (2 nM) and anti-MBG antibody. It is a Western blot showing the relative amount of.
Figure 2b shows the relative amount of protein kinase C-delta (PKC-δ, 78 kDa) in the nuclei of primary cardiac fibroblasts incubated with MBG alone (10 nM) or with MGB (10 nM) and anti-MBG antibody. It's Western Blot.
Figure 3a shows the proliferation level of human umbilical cord venous endothelial cells (HUVEC) incubated with MBG alone (2 nM) or MGB (2 nM) and human anti-MBG antibody (20 μg/mL) together. .
3B shows the proliferation level of human umbilical vein endothelial cells (HUVEC) incubated with MBG alone (2 nM) or MGB (2 nM) and human anti-MBG antibody (2.5-10 μg/mL).
4 shows the reading _{of OD 630} as a measure of Pi, which is a measure of ^{Na +} K ^{+ -ATPase activity.} The bar graph shows the effect of MBG on Pi levels, reversal of this effect by anti-MBG antibodies.
5 shows a standard curve for MBG in a competitive ELISA assay for measuring MBG in biological samples.

While various aspects of the present disclosure have been described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous modifications and variations, and changes and substitutions to the aspects described herein will be apparent to those skilled in the art without departing from this disclosure. It is understood that various alternatives to the aspects described herein may be applied in practicing the present disclosure. In addition, it is understood that all aspects of the present disclosure may be optionally combined with any one or more other aspects described herein consistent with those aspects.

When presenting elements in list form (eg, Markush group), it is understood that each possible subgroup of elements is also disclosed, and that any one or more elements may be removed from the list or group.

Conversely, unless expressly indicated to the contrary, in any method described or claimed herein that includes one or more acts or steps, the order of acts or steps of the method is not necessarily limited to the order in which the acts or steps of the method are mentioned. In other words, it is understood that the present disclosure includes aspects in which the order is so limited.

In general, where an aspect of the detailed description or claims is stated to include one or more features, it is also understood that the present disclosure also includes aspects consisting of or consisting essentially of such feature(s).

It is further understood that any aspect of the present disclosure, for example any aspect found within the prior art, may be expressly excluded from the claims, regardless of whether a particular exclusion is recited in the specification.

Reference herein to a peptide, polypeptide or protein, such as an antibody or fragment thereof, is also understood to include pharmaceutically acceptable salts thereof, unless specifically stated otherwise or the context dictates otherwise. Such salts may have a positive net charge, a negative net charge, or a no net charge.

Headings are incorporated herein for reference and to assist in finding specific sections. The headings are not intended to limit the scope of the aspects and concepts described in the sections below that heading, and such aspects and concepts may be applied in other sections throughout the entire disclosure.

All patent documents and all non-patent documents cited herein are incorporated herein by reference in their entirety to the same extent as if each patent document or non-patent document was specifically and individually indicated to be incorporated herein by reference in its entirety. do.

I. Definition

Unless otherwise defined or otherwise clearly indicated by use herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As used in the specification and appended claims, the indefinite articles "a" and "an" and the definite article "the" include the singular as well as the plural referent unless specifically stated otherwise or the context dictates otherwise. can do.

The term “about” or “approximately” means an acceptable error for a particular value as determined by a person skilled in the art that depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1 standard deviation. In some embodiments, where a specific margin of error (eg, standard deviation of the mean value provided in a chart or data table) is not stated, the term “about” or “approximately” refers to the range inclusive of the recited value and significant digits. It means the range included by rounding or rounding to the value quoted in consideration. In certain embodiments, the term “about” or “approximately” means within ±10%, 5%, 4%, 3%, 2% or 1% of the specified value. Whenever the term "about" or "approximately" precedes the first numeric value in a series of two or more numeric values or a range of two or more numeric values, the term "about" or "approximately" means a series of numeric values or a series of numbers Applies to each of the numeric values in the value range.

The term "antibody" is a protein that is functionally defined as a binding protein and structurally defined as comprising an amino acid sequence recognized as derived from the framework region of an immunoglobulin encoding gene. Say. An antibody may consist of one or more polypeptides that are substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, and in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.

It is known that the general gamma immunoglobulin (antibody) structural unit contains a tetramer. Each tetramer consists of a pair of two identical polypeptide chains, each pair having one “light chain” (about 25 kD) and one “heavy chain” (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to such light and heavy chains, respectively.

Antibodies exist as intact immunoglobulins or as several well characterized fragments. Thus, for example, pepsin is a dimer of Fab', a light chain linked to the VH-CH1-hinge naturally by disulfide bonds by digestion of the antibody under the disulfide bond in the hinge region. (ab)' produces ₂ F(ab)' ₂ can be reduced under mild conditions to break the disulfide bond(s) in the hinge region _{to convert the F(ab)' 2} dimer to the Fab' monomer. The Fab' monomer is essentially a Fab that has part of the hinge region (see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of digestion of intact antibodies, one of skill in the art will recognize that fragments can be synthesized chemically or in novel ways using recombinant DNA methods. Thus, the term antibody as used herein also includes antibody fragments produced by modification of whole antibodies or synthesized using recombinant DNA methodology. _{Preferred antibodies are V H} -V _L dimers, including single chain antibodies (antibodies present as single polypeptide chains), for example single chain Fv antibodies (sFv or scFv) with variable heavy and variable light chain regions bound together (direct or peptide linker Through) to form a contiguous polypeptide. _{Single chain Fv antibodies are covalently linked V H} -V _L heterodimers that can be expressed from nucleic acids comprising _{V H} -and V _L -encoding sequences that are directly linked or linked by a peptide-encoding linker (e.g. , Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988, which is hereby incorporated by reference in its entirety). V _H and V _L are each linked by a single polypeptide chain, but the V _H and V _L domains are non-covalently linked. Alternatively, the antibody may be another fragment. Other fragments can also be produced, including using recombinant techniques. For example, if one of the chains (heavy chain or light chain) is fused to a g3 capsid protein, and the complementary chain is exported to the periplasm as a soluble molecule, the Fab molecule can appear on the phage. The two chains can be encoded on the same or different replicons: in each Fab molecule the two antibody chains are assembled post-translationally, and the dimer is integrated into the phage particle via linkage to one of the g3p chains. (See, for example, U.S. Patent No. 5,733,743, which is incorporated herein by reference in its entirety). ScFv antibodies and many other structures are known to those skilled in the art that convert naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into molecules that fold into a three-dimensional structure substantially similar to that of the antigen binding site (e.g. See, for example, U.S. Patent Nos., 091,513, 5,132,405, and 4,956,778, which are incorporated herein by reference in their entirety). Particularly preferred antibodies include all those produced by recombinant techniques using vectors that appear on phage or whose chains are secreted into soluble proteins, for example scFv, Fv, Fab, (Fab') _2. In addition, the antibody may include a diabody and a minibody.

In addition, antibodies include heavy chain dimers such as camel antibodies. _{Since the V H} region of the heavy chain dimer IgG of camels does not have to have a hydrophobic interaction with the light chain, the region of the heavy chain in contact with the light chain is generally changed to a hydrophilic amino acid residue of the camel. _{The V H} domain of heavy chain dimer IgG is referred to as the V _{HH domain.}

In camels, the diversity of the antibody repertoire is determined by complementarity determining regions (CDRs) 1, 2 and 3 in the _{V H} or V _{HH regions.} The CDR3 of the camel V _HH region is characterized by a relatively long length of an average of 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129, the entire contents of which are incorporated by reference). This contrasts with the CDR3 regions of many other species of antibodies. For example, _{the CDR3 of mouse V H} has an average of 9 amino acids.

Libraries of camel-derived antibody variable regions that maintain the in vivo diversity of camel variable regions are, for example, in the method disclosed in U.S. Patent Application Publication No. US20050037421 published on February 17, 2005, the contents of which are incorporated by reference in their entirety. Can be manufactured by

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of antibody action. ADCC can be improved by several methods, many of which relate to terminal product antibodies with improved Fc-receptor binding. Amino acid substitutions in the antibody Fc region increase the Fc binding affinity for the FcRIIIa receptor on NK cells (Natsume et al., Drug Design, Development and Therapy, 2009, vol. 3, pp. 7-16, which is hereby incorporated by reference in its entirety), has been shown to improve ADCC activity. Another way to improve ADCC is to alter the sugar composition of antibody glucosylation. This is done by generating antibodies that lack fucose residues, ie'a-fucosylated' or'de-fucosylated' antibodies. One method involves altering/modifying the glycosylation site of the antibody so that fucose cannot be added to the antibody (patent: 6194551, February 27, 2001). Another method is to remove fucose already present on the antibody, for example by removal of fucose or enzymatic digestion by any other means. Other methods include genetic engineering of host expression systems such that fucose cannot be transferred to or added to the antibody by, for example, inhibition or deletion of the fucosyl transferase activity (US Patent Application Publication No. 20070134759, 20017 6 May 14; U.S. Patent Application Publication 20080166756, July 10, 2008, all of which are incorporated herein by reference in their entirety).

As used herein, the term "antigen" is a substance capable of inducing a specific immune response under appropriate conditions and reacting with a product of that reaction, such as a specific antibody or specifically sensitized T-lymphocyte or both. Say. Antigens may be soluble substances such as toxins and foreign proteins, or may be particulates such as bacterial and tissue cells; Only a portion of a protein or polysaccharide molecule known as an antigenic determinant (epitope) is bound to an antibody or a specific receptor on a lymphocyte. More broadly, the term “antigen” can be used to refer to any substance to which an antibody binds or for which an antibody is required, whether or not the substance is immunogenic. For these antigens, antibodies can be identified by recombinant methods regardless of any immune response.

As used herein, the term "binding specificity" of an antibody refers to the identity of the antigen to which the antibody binds, preferably the identity of the epitope to which the antibody binds.

As used herein, the term "chimeric polynucleotide" means that the polynucleotide includes a wild-type region and a mutated region. Further, this may mean that the polynucleotide comprises a wild-type region from one polynucleotide and a wild-type region from another related polynucleotide.

As used herein, the term "complementarity-determining region" or "CDR" refers to a term recognized in the art illustrated by Kabat and Chothia. In addition, CDRs are generally known as hypervariable regions or hypervariable loops (Chothia and Lesk (1987) J Mol. Biol. 196: 901; Chothia et al. (1989) Nature 342: 877; EA Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) (1987); and Tramontano et al. (1990) J Mol. Biol. 215: 175, all of which are here Is incorporated by reference in its entirety). "Framework region" or "FR" refers to the region of the V domain that franks the CDRs. The location of the CDR and framework regions can be determined using various well-known definitions in the art, for example Kabat, Chothia, the international ImMunoGeneTics database (IMGT) and AbM (eg, Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions.Nature 342, 877-883; Chothia See C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)). In addition, the definition of the antigen binding site is described below: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); And Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); And Rees et al, In Sternberg M. J. E. (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996, all of which are incorporated herein by reference in their entirety.

Whenever the term "at least" or "greater than" precedes the first numerical value in a series of two or more numerical values, the term "at least" or "greater than" means each of the numerical values in the series of numerical values. Applies to

The term “heterologous” refers to an amino acid or nucleotide sequence that is not naturally found in association with a linked amino acid or nucleotide sequence.

The terms "natural" or "naturally occurring" as applied to an object refer to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including a virus), can be isolated from natural sources, and has not been intentionally modified by humans in the laboratory, is a naturally occurring one.

Whenever the term "no more than" or "less than" precedes the first numeric value in a series of two or more numeric values, the term "less than" or "less than" means the numeric values in the series of numeric values. It applies to each.

The term "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include non-naturally occurring bases, nucleotides involved in linkage with nucleotides other than naturally occurring phosphodiester linkages, or/and those containing bases attached through linkages other than phosphodiester linkages. . Non-limiting examples of nucleotide analogues include phosphorothioate, phosphorodithioate, phosphorotriester, phosphoramidate, boranophosphate, methylphosphate. Phonate (methylphosphonate), chiral-methyl phosphonate (chiral-methyl phosphonate), 2-O-methyl ribonucleotide (2-O-methyl ribonucleotide), peptide-nucleic acid (PNA), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “nucleic acid molecule” generally refers to a larger polynucleotide. The term “oligonucleotide” generally refers to a shorter polynucleotide. In certain embodiments, the oligonucleotide contains no more than about 50 nucleotides. When the nucleotide sequence is represented by a DNA sequence (ie A, T, G, C), it is understood to include an RNA sequence (ie A, U, G, C) where “U” is replaced by “T” .

The term "polypeptide" is composed of natural and/or non-natural amino acid residues, naturally occurring structural variations thereof, and/or synthetic and non-naturally occurring analogs thereof that are bound through peptide bonds. Refers to the polymer. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. In addition, the polypeptide can be produced recombinantly in cells that display the nucleic acid sequence encoding the polypeptide. The term “protein” generally refers to a larger polypeptide. The term “peptide” generally refers to a shorter polypeptide. In certain embodiments, the peptide contains no more than about 50, 40 or 30 amino acid residues. Polypeptides include antibodies and fragments thereof. Conventional notation is used herein to describe a polypeptide sequence: the left end of the polypeptide sequence is the amino (N)-terminus; The right end of the polypeptide sequence is the carboxyl (C)-terminus.

A polypeptide may contain one or more modifications that can be made during the course of synthesis or cellular production of the polypeptide, such as one or more post-translational modifications, whether one or more modifications are intentional or not. Modifications may be, without limitation, glycosylation (e.g., N-linked glycosylation and O-linked glycosylation), lipidation, phosphorylation, sulfurization, acetylation (e.g., N-terminal acetylation), amidation (e.g. For example, C-terminal amidation), hydroxylation, methylation, intramolecular or intermolecular disulfide bond formation, lactam formation between two side chains, pyroglutamate formation and ubiquitination. As another example, the polypeptide may be a natural polymer (eg polysaccharide) or a synthetic polymer (eg polyethylene glycol [PEG]), lipidated (eg, C ₈ -C ₂₀ acyl acylated), Or labeled with a detectable agent (eg, radionuclides, fluorescent dyes or enzymes). PEGylation can increase protease resistance, stability and half-life, increase solubility, and reduce aggregation of polypeptides.

The term “conservative substitution” refers to the substitution of amino acids in a polypeptide with natural or non-natural amino acids that are functionally, structurally or chemically similar. In certain embodiments, the following groups each contain natural amino acids that are conservative substitutions for each other:

1) glycine (Gly/G), aniline (Ala/A);

2) isoleucine (Ile/I), leucine (Leu/L), methionine (Met/M), valine (Val/V);

3) phenylalanine (Phe/F), tyrosine (Tyr/Y), tryptophan (Trp/W);

4) serine (Ser/S), threonine (Thr/T), cysteine (Cys/C);

5) Asparagine (Asn/N), glutamine (Gln/Q);

6) Aspartic acid (Asp/D), glutamic acid (Glu/E); And

7) Arginine (Arg/R), lysine (Lys/K), histidine (His/H).

In another embodiment, the following groups each contain natural amino acids that are conservative substitutions for each other:

1) Non-polar: Ala, Val, Leu, Ile, Met, Pro (Proline/P), Phe, Trp;

2) hydrophobicity: Val, Leu, Ile, Phe, Tyr, Trp;

3) aliphatic: Ala, Val, Leu, Ile;

4) aromatic: Phe, Tyr, Trp, His;

5) uncharged polar or hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gin, Tyr (tyrosine can be considered a hydrophobic amino acid with polar side groups);

6) aliphatic hydroxyl- or sulfhydryl-containing: Ser, Thr, Cys;

7) amide-containing: Asn, Gin;

8) acidic: Asp, Glu;

9) Basic: Lys, Arg, His; And

10) Small: Gly, Ala, Ser, Cys.

In other embodiments, amino acids can be grouped as shown below:

1) Hydrophobicity: Val, Leu, Ile, Met, Phe, Trp, Tyr;

2) aromatic: Phe, Tyr, Trp, His;

3) neutral hydrophilicity: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gin;

4) acidic: Asp, Glu;

5) Basic: Lys, Arg, His; And

6) Residues that influence backbone orientation: Pro, Gly.

A polypeptide having one or more modifications relative to the parent polypeptide may suitably be referred to as a “analog”, “derivative” or “variant” of the parent polypeptide.

The present disclosure includes pharmaceutically acceptable salts of polypeptides including those with positive net charge, negative net charge or no net charge.

The term “pharmaceutically acceptable” is suitable for use in contact with tissues and organs of a subject without excessive irritation, allergic reactions, immunogenicity and toxicity, corresponds to a reasonable benefit/risk ratio, and intended use. It refers to a substance (for example, an active ingredient or excipient) that is effective for In addition, the “pharmaceutically acceptable” excipient or carrier of the pharmaceutical composition may be compatible with the other components of the composition.

The term "stringent hybridization condition" refers to hybridization with 50% formamide at 5XSSC at 42°C and washing the filter at 0.2XSSC at 60°C (1XSSC is 0.15M NaCl, 0.015M sodium sheet Rate). In addition, stringent hybridization conditions include, for example, washing 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; Denaturing agents such as formamide, for example 750 mM sodium chloride, 75 mM sodium citrate at 42° C. and 50% (v/v) formamide at pH 6.5 and 0.1% bovine serum albumin/0.1% Ficoll/0.1 Hybridized with% polyvinylpyrrolidone/50 mM sodium phosphate buffer; Or 50% formamide, 5XSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt solution, sonicated salmon sperm DNA (50 μg /ml), 0.1% SDS, and hybridized with 10% dextran sulfate at 42° C., washed at 55° C. in 0.2XSSC (sodium chloride/sodium citrate) and 50% formamide at 42° C., and then at 55° C. Includes those with low ionic strength and high temperatures for very rigorous cleaning consisting of 0.1XSSC containing EDTA.

The term “subject” refers to primates (eg, humans, chimpanzees or monkeys), rodents (eg, rats, mice, guinea pigs, gerbils or hamsters), and order of rabbits (eg, rabbits). , Pigs (for example, pigs), horses (for example, horses), canines (for example, dogs) or cats (for example, cats), including, but not limited to, animals .

The terms “substantially homologous” or “substantially identical” in the context of two polypeptides or polynucleotides are compared for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. And at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid or nucleic acid residue identity when aligned. It refers to two or more sequences or subsequences having. The terms “substantially homologous” or “substantially identical” can mean at least about 70% amino acid or nucleic acid residue identity. The terms “substantially homologous” or “substantially identical” can mean at least about 85% amino acid or nucleic acid residue identity. In fact, homology or identity may exist on a region of the sequence that is at least about 20, 30, 40, 50, 100, 150 or 200 residues in length. The sequences may be substantially homologous or identical over the entire length of each or all of the comparative biological polymers.

Optimal alignment of sequences for comparison is described, for example, in the local homology algorithm of Smith and Waterman, Adv. Appl. Math. , 2 :482 (1981); the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol ., 48 :443 (1970); the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA , 85 :2444 (1988); By computerized implementations of these algorithms (eg, Wisconsin, Madison, Genetics Computer Group, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package); Or it can be carried out by visual inspection.

One example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments in groups of related sequences using progressive pair alignments to indicate percent relationship and sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP is Feng and Doolittle, J. Mol. Evol ., 35 :351-360 (1987) uses a simplification of the progressive alignment method. The method used is similar to the method described in Higgins and Sharp, CABIOS , 5:151-153 (1989). The program can align up to about 300 sequences, each up to about 5,000 nucleotides or amino acids in length. The multiple alignment procedure starts with a pair alignment of the two most similar sequences, creating a cluster of two aligned sequences. This cluster is then aligned to the next most relevant sequence or cluster of aligned sequences. The clusters of two sequences are aligned by a simple extension of the pairwise alignment of two separate sequences. The final alignment is achieved by a series of progressive pair alignments. The program is executed by specifying a specific sequence and its amino acid or nucleotide coordinates for the sequence comparison region, and by specifying program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and Weighted end gap. Another algorithm useful for generating multiple alignments of sequences is Clustal W (see, eg, Thompson et al. , Nucleic Acids Research , 22 :4673-4680 [1994]).

Other examples of algorithms suitable for determining sequence identity and percent sequence similarity are described in Altschul et al ., J. Mol. Biol ., 215 :403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. These algorithms involve identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence, which when aligned with words of the same length in the database sequence. It matches or satisfies the positive-valued threshold score T. T is called a neighborhood word score threshold (Altschul 1990). These initial adjacent word hits start searching and serve as seeds containing longer HSPs. Thereafter, as long as the cumulative alignment score can increase, the word hits are expanded in both directions along each sequence. The cumulative score is calculated using the parameters M (reward score for a matched pair of residues, always >0) and N (penalty score for mismatched residues, always <0) for nucleotide sequences. For amino acid sequences, a score matrix is used to calculate the cumulative score. In the following cases, the expansion of word hits in each direction is stopped: the cumulative alignment score falls from the maximum achieved value by the quantity X; The cumulative score goes below zero due to the accumulation of one or more negative scoring residue alignments; Alternatively, the end of both sequences is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults, for example word length (W) 11, expected value (E) 10, M = 5, N = -4, and a comparison of two strands. For amino acid sequences, the BLASTP program uses, for example, word length (W) 3, expected value (E) 10, and BLOSUM62 scoring matrix as defaults (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA , 89 :10915 [1989]).

In addition to calculating the percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (eg, Karlin and Altschul, Proc. Natl. Acad. Sci. USA , 90 :5873-5787 [199393] ] Reference). One measure of similarity provided by the BLAST algorithm is the minimum sum probability [P(N)], which indicates the probability that a match between two nucleotide or amino acid sequences will occur by chance. In certain embodiments, a polynucleotide is considered to be similar to a reference sequence if the minimum sum probability in comparison of a test polynucleotide and a reference polynucleotide is less than about 0.1, 0.01, or 0.001.

In some embodiments, the polypeptide is substantially homologous or identical to the second polypeptide when the two polypeptides differ only by conservative amino acid substitutions. In certain embodiments, as described herein, when two polynucleotides hybridize to each other under stringent conditions or under very stringent conditions, the two nucleic acid sequences are substantially homologous or identical.

The term "therapeutically effective amount", when administered to a subject, prevents, reduces, delays the onset, slows the progression of the risk of developing, causes a regression of the medical condition being treated, or causes a medical condition or one of those conditions. It refers to the amount of the compound sufficient to alleviate the above symptoms or complications to some extent. In addition, the term "therapeutically effective amount" refers to an amount of a compound sufficient to elicit a biological or medical response in a cell, tissue, organ, system, animal or human being sought by a researcher, veterinarian, physician or clinician.

The terms “treat”, “treating” and “treatment” alleviate, ameliorate or abolish one or more symptoms or complications associated with a medical condition or condition, and alleviate one or more causes of the condition. , To improve or eradicate. Reference to “treatment” of a medical condition includes prevention of the condition. The terms “prevent”, “preventing” and “prevention” include preventing, reducing and delaying the onset of the medical condition or one or more symptoms or complications associated with that condition.

II. Anti-Marinobufagenin Antibody

The antibodies described herein have specificity for marinobufagenin (MBG) and include all of the forms described above. Antibodies can be engineered for use in specific organs. Organs may be humans, dogs, or commercially valuable livestock such as, for example, pigs, horses, dogs, cats, chickens, or other birds. Such manipulation of antibodies can be performed using, for example, any repertoire techniques or monoclonal techniques known in the art, humanization, humaneering, chimerization, or human (or other organisms). Includes the isolation of antibodies.

Affinity maturation can be used in conjunction with the antibodies disclosed herein to obtain anti-MBG antibodies of the desired affinity. When anti-MBG antibodies are obtained from an animal (eg, a transgenic animal having a human antibody repertoire), antibodies made in the transgenic animal can undergo affinity maturation. Alternatively, antibodies from transgenic animals or antibodies from other technologies (e.g., display technologies) are subjected to affinity maturation using a chain shuffling approach and/or mutations of nucleic acids encoding VH and VL, Antibodies with greater affinity can be selected and/or selected.

The most widely used method to minimize the immunogenicity of a non-human antibody while maintaining specificity and affinity involves grafting the CDRs of a non-human antibody into a human framework generally selected for structural homology to the non-human framework ( Jones et al., 1986, Nature 321:522-5; US Pat. No. 5,225,539, all of which are incorporated herein by reference). Originally, this method has a sharp decline in affinity. However, it has been found that some of the affinity can be restored by restoring non-human residues at key positions in the framework necessary to maintain the standard structure of non-human CDRs 1 and 2 (Bajorath et al., 1995, J Biol Chem 270:22081 -4; Martin et al., 1991, Methods Enzymol. 203:121-53; Al-Lazikani, 1997, J Mol Biol 273:927-48, all of which are incorporated herein by reference in their entirety). Restoring the basic form of CDR3 is a much more uncertain task, as their structure is more diverse.

Improvements to traditional CDR-grafting approaches use a variety of hybrid selection approaches, where some of the non-human antibodies have been combined with a library of complementary human antibody sequences in successive rounds of selection for antigen binding, in the process most Non-human sequences are gradually replaced by human sequences. Another methodology involves a chain replacement technique that is individually replaced at a stage in which the non-human CDR3 is maintained and only the framework and the remaining V-regions including CDRs 1 and 2 are performed.

These techniques can be used to make antibodies suitable for use in non-human subjects by manipulating the CDRs into the framework regions of the species of interest using a similar approach to the CDR grafting method used to make antibodies for use in humans. .

The chimeric anti-MBG antibody can be made by using a starting anti-MBG antibody and grafting the variable region of the starting anti-MBG antibody to a variable domain constant region of interest. For example, chimeric antibodies were prepared by binding mouse VL and VH regions with human constant region CL (light chain constant region) and at least CH1 (heavy chain constant region 1). The VH regions used for chimeric antibodies are:

EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGTTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARARYGYGFDYWGQGTTLTVSS (SEQ ID NO: 1)

The VL regions used for chimeric antibodies are:

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNRNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK (SEQ ID NO: 2)

Anti-MBG, humanized antibodies are prepared using, for example, CDRs:

VH CDR1: DSITSGYWN (SEQ ID NO: 3)

VH CDR2: YISYSGTTYYNPSLKS (SEQ ID NO: 4)

VH CDR3: ARYGYGFDY (SEQ ID NO: 5)

V _L CDR1: RSSQSIVHSNRNTYLE (SEQ ID NO: 6)

V _L CDR2: KVSNRFS (SEQ ID NO: 7)

V _L CDR3: FQGSHVPLT (SEQ ID NO: 8)

The three VH CDRs are placed in the framework sequence of the human variable region for the heavy chain to generate the VH chain:

QVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRQPPGKGLEWIGYISYSGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARYGYGFDYWGQGTLVTVSS (SEQ ID NO: 9)

This VH is designated H1. Substitutions are introduced in the framework region of SEQ ID NO: 9 to create two additional VH chains:

EVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRQPPGKGLEYIGYISYSGTTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARARYGYGFDYWGQGTLVTVSS (SEQ ID NO: 10)

EVQLQESGPGLVKPSETLSLTCTVSGDSITSGYWNWIRQPPGKGLEYMGYISYSGTTYYNPSLKSRITISRDTSKNQYSLKLSSVTAADTAVYYCARARYGYGFDYWGQGTLVTVSS (SEQ ID NO: 11)

SEQ ID NO: 10 is designated as H2, and SEQ ID NO: 11 is designated as H3.

The three VL CDRs are placed in the framework sequence for the human variable region from the light chain to generate the VL chain:

DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNRNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGGGTKVEIK (SEQ ID NO: 12)

SEQ ID NO: 12 is designated as L1. Substitutions are introduced into the framework region of SEQ ID NO: 12 to create an additional VL chain:

DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNRNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGGGTKVEIK (SEQ ID NO: 13)

SEQ ID NO: 13 is designated L2. The VL chain SEQ ID NO: 12 is combined with each of the HL chains (SEQ ID NOs: 9, 10, and 11) to generate three different antibodies (H1L1, H2L1, and H3L1). In addition, the VL chain SEQ ID NO: 13 is combined with each of the HL chains (SEQ ID NOs: 9, 10, and 11) to generate three different antibodies (H1L2, H2L2, and H3L2).

Human, anti-MBG antibodies were obtained from human display libraries. 14 anti-MBG antibodies were obtained, and the sequences of these 14 human antibody heavy and light chains are as follows: The human antibody designated 236/237 has VH and VL sequences:

236/237 VH

QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARYNWNYVLFDYWGQGTLVTVSS (SEQ ID NO:14)

236/237 VL

QAVLTQPSSVSAAPRQRVTISCSGSASNIGRNGVSWYHQVPGKAPRLLLSHDGLVTSGVPDRLSVSKSGTSASLAISGLHSDDEGDYYCAVWDDSLNAVVFGGGTKLTVLS (SEQ ID NO: 15)

The VH CDR sequences for the 236/237 VH are:

236/237 VH CDR1 GGSISSSSYY (SEQ ID NO: 16)

236/237 VH CDR2 IYYSGST (SEQ ID NO: 17)

236/237 VH CDR3 ARARYNWNYVLFDY (SEQ ID NO: 18)

The VL CDR sequences for the 236/237 VL are:

236/237 VL CDR1 ASNIGRNG (SEQ ID NO: 19)

236/237 VL CDR2 HDG (SEQ ID NO: 20)

236/237 VL CDR3 AVWDDSLNAVV (SEQ ID NO: 21)

The human antibody designated 201/202 has VH and VL sequences:

201/202 VH

QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWGWIRQPPGKGLEWIGSIYYSGNTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARASIAARTFDYWGQGTLVTVSS (SEQ ID NO:22)

201/202 VL

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGTGTKVTVL (SEQ ID NO: 23)

The VH CDR sequences for the 201/202 VH are:

201/202 VH CDR1 GGSISSGGYY (SEQ ID NO: 24)

201/202 VH CDR2 IYYSGNT (SEQ ID NO: 25)

201/202 VH CDR3 ARASIAARTFDY (SEQ ID NO: 26)

The VL CDR sequences for the 201/202 VL are:

201/202 VL CDR1 SSNIGNNY (SEQ ID NO: 27)

201/202 VL CDR2 DNN (SEQ ID NO: 28)

201/202 VL CDR3 GTWDSSLSAYV (SEQ ID NO: 29)

Human antibodies designated 203/204 have VH and VL sequences:

203/204 VH

QLQLQESGPGLVKPSETLSLTCTVSGGSISSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLQLKSVTPEDTAVYYCARVRPPAATFDYWGQGTLVTVSS (SEQ ID NO:30)

203/204 VL

QSVVTQPPSVSAAPGQKVSISCSGGSSNIGNNYVSWYQQLPGTAPRLLIYENNNRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLRAVVFGGGTKLTVL (SEQ ID NO: 31)

The VH CDR sequences for the 203/204 VH are:

203/204 VH CDR1 GGSISSYY (SEQ ID NO: 32)

203/204 VH CDR2 IYYSGST (SEQ ID NO: 17)

203/204 VH CDR3 ARVRPPAATFDY (SEQ ID NO: 33)

The VL CDR sequences for the 203/204 VL are:

203/204 VL CDR1 SSNIGNNY (SEQ ID NO: 27)

203/204 VL CDR2 ENN (SEQ ID NO: 34)

203/204 VL CDR3 GTWDSSLRAVV (SEQ ID NO: 35)

Human antibodies designated 206/208 have VH and VL sequences:

206/208 VH

QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVNLRGAFDIWGQGTTVTVSS (SEQ ID NO:36)

206/208 VL

QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYENNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVL (SEQ ID NO: 37)

The VH CDR sequences for the 206/208 VH are:

206/208 VH CDR1 GGSISSSSYY (SEQ ID NO: 16)

206/208 VH CDR2 IYYSGST (SEQ ID NO: 17)

206/208 VH CDR3 ARVNLRGAFDI (SEQ ID NO: 38)

The VL CDR sequences for the 206/208 VL are:

206/208 VL CDR1 SSNIGAGYD (SEQ ID NO: 39)

206/208 VL CDR2 ENN (SEQ ID NO: 34)

206/208 VL CDR3 GTWDSSLSAVV (SEQ ID NO: 40)

Human antibodies designated 238/239 have VH and VL sequences:

238/239 VH

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVVVARTGTTISWFDPWGQGTLVTVSS (SEQ ID NO: 41)

238/239 VL

QAVLTQPSSLSASPGASASLTCTLRSGINVDTYRIYWYQQKPGSPPQYLLRYKSDSDKQQGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCVIWHSSAWVFGGGTKLTVL (SEQ ID NO: 42)

The VH CDR sequences for the 238/239 VH are:

238/239 VH CDR1 GGSISSSSYY (SEQ ID NO: 16)

238/239 VH CDR2 IYYSGST (SEQ ID NO: 17)

238/239 VH CDR3 ARVVVARTGTTISWFDP (SEQ ID NO: 43)

The VL CDR sequences for the 238/239 VL are:

238/239 VL CDR1 SGINVDTYR (SEQ ID NO: 44)

238/239 VL CDR2 YKSDSDK (SEQ ID NO: 45)

238/239 VL CDR3 VIWHSSAWV (SEQ ID NO: 46)

Human antibodies designated 240/241 have VH and VL sequences:

240/241 VH

QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLRYSSGWYADPWGQGTLVTVSS (SEQ ID NO: 47)

240/241 VL

LPVLTQPPSASGTPGQRVTISCSGSSSNIGSNIVNWYQQLPGTAPKLLIYNNNQRPSGVPDRFSGSKSGASASLAISGLQSEDEADYYCVAWDGSLSGVVFGGGTKLTVL (SEQ ID NO: 48)

The VH CDR sequences for the 240/241 VH are:

240/241 VH CDR1 GGSISSSSYY (SEQ ID NO: 16)

240/241 VH CDR2 IYYSGST (SEQ ID NO: 17)

240/241 VH CDR3 ARLRYSSGWYADP (SEQ ID NO: 49)

The VL CDR sequences for the 240/241 VL are:

240/241 VL CDR1 SSNIGSNI (SEQ ID NO: 50)

240/241 VL CDR2 NNN (SEQ ID NO: 51)

240/241 VL CDR3 VAWDGSLSGVV (SEQ ID NO: 52)

Human antibodies designated 205/207 have VH and VL sequences:

205/207 VH

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARFHTWAPRAFDIWGQGTMVTVSS (SEQ ID NO: 53)

205/207 VL

QSVVTQPPSVSAAPGQEVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSALFGGGTKLTVL (SEQ ID NO: 54)

The VH CDR sequences for the 205/207 VH are:

205/207 VH CDR1 GGSISSSSYY (SEQ ID NO: 16)

205/207 VH CDR2 IYYSGST (SEQ ID NO: 17)

205/207 VH CDR3 ARFHTWAPRAFDI (SEQ ID NO: 55)

The VL CDR sequences for the 205/207 VL are:

205/207 VL CDR1 SSNIGNNY (SEQ ID NO: 27)

205/207 VL CDR2 DNN (SEQ ID NO: 28)

205/207 VL CDR3 GTWDSSLSAL (SEQ ID NO: 56)

Human antibodies designated R4CE-1E have VH and VL sequences:

R4CE-1E VH

QVQLQESGSGLVKPSQTLSLTCTVSGGSISSSGYYWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVQLWQRGFDYWGQGTLVTVSS (SEQ ID NO: 57)

R4CE-1E VL

DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKINRVEAEDVGVYYCMQALQAPLTFGGGTKVEIK (SEQ ID NO: 58)

The VH CDR sequences for R4CE-1E VH are:

R4CE-1E 8 VH CDR1 GGSISSSGYY (SEQ ID NO: 59)

R4CE-1E 8 VH CDR2 IYHSGST (SEQ ID NO: 60)

R4CE-1E 8 VH CDR3 ARVQLWQRGFDY (SEQ ID NO: 61)

The VL CDR sequences for R4CE-1E VL are:

R4CE-1E 8 VL CDR1 QSLLHSNGYNY (SEQ ID NO: 62)

R4CE-1E 8 VL CDR2 LGS (SEQ ID NO: 63)

R4CE-1E 8 VL CDR3 MQALQAPLT (SEQ ID NO: 64)

Human antibodies designated 2A-F8 have VH and VL sequences:

2A-F8 VH

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVTWSDYRDYWGQGTLVTVSS (SEQ ID NO: 65)

2A-F8 VL

EIVMTQSPGTLSLSPGERATLSCRASQSVTSRRVAWFQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQGLSQSPVDKLTKVQWKVDNALKLSQDSQ

The VH CDRs for 2A-F8 VH are:

2A-F8 9 VH CDR1 GGSFSGYY (SEQ ID NO: 67)

2A-F8 9 VH CDR2 INHSGST (SEQ ID NO: 68)

2A-F8 9 VH CDR3 VTWSDYRDY (SEQ ID NO: 69)

The VL CDR sequences for the 2A-F8 VL are:

2A-F8 9 VL CDR1 QSVTSRR (SEQ ID NO: 70)

2A-F8 9 VL CDR2 GAS (SEQ ID NO: 71)

2A-F8 9 VL CDR3 QQYGSSPLT (SEQ ID NO: 72)

Human antibodies designated R4CE-7G have VH and VL sequences:

R4CE-7G VH

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGGTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTTYSYGPGFDPWGQGTLVTVSSASTKG (SEQ ID NO: 73)

R4CE-7G VL

QSVVTQPPSVSAAPGQKVTISCSGSSSNIGTYYVSWYQQFPGTVPKLLIYDNTQRPSEIPDRFSASKSGTSVTLDITGLQTGDEGDYYCATWDSNLKSVVFGGGTKVAVLGQPKAAPSVTLFPPSSEELQSNAPKATLVCCLISSKGQSNAPKASSYCLISSKGDFGSNAPASTVCLISSKGDFGA

The VH CDR sequences for R4CE-7G are:

R4CE-7G 10 VH CDR1 GGSISSSSYY (SEQ ID NO: 16)

R4CE-7G 10 VH CDR2 IYYSGGT (SEQ ID NO: 75)

R4CE-7G 10 VH CDR3 ARTTYSYGPGFDP (SEQ ID NO: 76)

The VL CDRs for R4CE-7G are:

R4CE-7G 10 VL CDR1 SSNIGTYY (SEQ ID NO: 77)

R4CE-7G 10 VL CDR2 DNT (SEQ ID NO: 78)

R4CE-7G 10 VL CDR3 ATWDSNLKSVV (SEQ ID NO: 79)

Human antibodies designated R4CE-10H have VH and VL sequences:

R4CE-10H VH

QLQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGSIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVMYYYGSGSPYWYFDLWGRGTLVTVSS (SEQ ID NO: 80)

R4CE-10H VL

QSVVTQPPSVSAAPGQKVTISCSGSSSNIGKNFVSWYQQFPGTAPKFLIYENSKRPSGIPDRISGSKSGTSATLAITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISGSSSNIGKNFVSWYQQFPGTAPKFLIYENSKRPSGIPDRISGSKSGTSATLAITGLQTGDEADYYCGTWDSSLSAVVFGGGTKLTVLSQPKAAPSVTLFPPSSEELQANKATLVCLISSNDFYPGAVSYVESK

The VH CDR sequences for R4CE-10H are:

R4CE-10H 11 VH CDR1 GGSISSGGYY (SEQ ID NO: 82)

R4CE-10H 11 VH CDR2 IYHSGST (SEQ ID NO: 83)

R4CE-10H 11 VH CDR3 ARVMYYYGSGSPYWYFDL (SEQ ID NO: 84)

The VL CDR sequences for R4CE-10H are:

R4CE-10H 11 VL CDR1 SSNIGKNF (SEQ ID NO: 85)

R4CE-10H 11 VL CDR2 ENS (SEQ ID NO: 86)

R4CE-10H 11 VL CDR3 GTWDSSLSAVV (SEQ ID NO: 40)

Human antibodies designated 1B-6C have VH and VL sequences:

1B-6C VH

QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISLDTSKNQFSLKLSSLTAADTAVYYCARVPAIRSSAIFDYWGQGTLVTVSS (SEQ ID NO: 87)

1B-6C VL

QSVVTQPPSVSAAPGQKVTISCFGSRSNIGNNYVSWYQQFPGTAPQLLIFDNNKRPSGIPDRFSGSKSGTSASLGITGVQTGDEAEYYCGAWDSSLSAGAVFGGGTKLTVLGQSPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAKASSYVELTQVDSKVKVKASTV

The VH CDR sequences for 1B-6C are:

1B-6C-12 VH CDR1 GGSISSSSYY (SEQ ID NO: 16)

1B-6C-12 VH CDR2 IYYSGST (SEQ ID NO: 17)

1B-6C-12 VH CDR3 ARVPAIRSSAIFDY (SEQ ID NO: 89)

The VL CDR sequences for 1B-6C are:

1B-6C-12 VL CDR1 RSNIGNNY (SEQ ID NO: 90)

1B-6C-12 VL CDR2 DNN (SEQ ID NO: 28)

1B-6C-12 VL CDR3 GAWDSSLSAGAV (SEQ ID NO: 91)

Human antibodies designated 1B-6E have VH and VL sequences:

1B-6E-13 VH

QVQLQESGPGLVKPSETLSLTCTVSGYSISSGYYWGWIRQPPGKGLEWIGSIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVAPYSSSPSFDYWGQGTLVTVSS (SEQ ID NO: 92)

1B-6E VL

LPVLTQPPSASGTPGQRVTISCSGSSSNIGRNTGNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVVFGGGTKVTVLGQSPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKASSYVELTCSVYPGAVKASSYTV

The VH CDRs for 1B-6E are:

1B-6E-13 VH CDR1 GYSISSGYY (SEQ ID NO: 94)

1B-6E-13 VH CDR2 IYHSGST (SEQ ID NO: 95)

1B-6E-13 VH CDR3 ARVAPYSSSPSFDY (SEQ ID NO: 96)

The VL CDR sequences for 1B-6E are:

1B-6E-13 VL CDR1 SSNIGRNT (SEQ ID NO: 97)

1B-6E-13 VL CDR2 SNN (SEQ ID NO: 98)

1B-6E-13 VL CDR3 AAWDDSLNGVV (SEQ ID NO: 99)

Human antibodies designated 2A-5A have VH and VL sequences:

2A-5A VH

QVQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARFSRGGPFLWGRGTLVTVSS (SEQ ID NO: 100)

2A-5A VL

QSVVTQPPSVSAAPGQKVTISCSGSSSNIGSYSVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAYVFGSGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPSPGAKVGSTVTLAPASSYCSPKQVNKAGVELTASKPSKVKWTV

The VH CDR sequences for 2A-5A are:

2A-5A-14 VH CDR1 GGSISSSSYY (SEQ ID NO: 16)

2A-5A-14 VH CDR2 IYYSGST (SEQ ID NO: 17)

2A-5A-14 VH CDR3 ARFSRGGPFL (SEQ ID NO: 102)

The VL CDR sequences for 2A-5A are:

2A-5A-14 VL CDR1 SSNIGSYS (SEQ ID NO: 103)

2A-5A-14 VL CDR2 DNN (SEQ ID NO: 28)

2A-5A-14 VL CDR3 GTWDSSLSAYV (SEQ ID NO: 29)

The 8 human VH regions have the same CDR1 of GGSISSSSYY (SEQ ID NO: 16). The 8 human VH regions have the same CDR2 of IYYSGST (SEQ ID NO: 17). Four of the human VL regions have the same CDR2 of DNN (SEQ ID NO: 28). Three of the human VL regions have the same CDR1 of SSNIGNNY (SEQ ID NO: 27). The CDR sequences of a human antibody can be grafted into the framework regions of the antibody from other species. In addition, the human CDR sequences can be mixed and matched in a combinatorial manner to make a new anti-MBG antibody. In addition, human anti-MBG antibodies are affinity matured using, for example, concise mutagenesis (e.g., Balint and Larrick, 1993) to obtain anti-MBG antibodies having a greater binding affinity for MBG. can do.

Any of the anti-MBG antibodies disclosed herein are, for example, antibody fragments (Fab, Fab', F(ab') ₂ , F _v , and other degradation fragments, as well as recombinant fragments, such as scFv, diabody ( diabody), triabody, tetrabody, Bis-scFv, minibody, Fab2, or Fab3) or full length antibodies such as IgA, IgD, IgE, IgG and IgM antibody). The full length IgA can be IgA1 or IgA2.

The present disclosure includes, without limitation, positive net charge, negative net charge, non-net charge, and in pharmaceutical compositions, therapeutic and diagnostic uses, in their production, anti-MBGs, including fragments thereof as compounds It includes pharmaceutically acceptable salts of anti-MBG antibodies, including salts of antibodies.

III. Anti-MBG antibody modification

Anti-MBG antibodies may _{comprise moieties that extend the half-} life (T 1/2) or/and duration of action of the antibody fragment. The moiety is circulating T _1/2 of antibody, blood T _1/2 , plasma T _1/2 , serum T _1/2 , terminal T _1/2 , biological T _1/2 , elimination T _1/2 or functional T _{1 /2} , or any combination thereof.

In certain embodiments, the anti-MBG antibody fragment comprises a single moiety. In other embodiments, the anti-MBG antibody fragment comprises two or more substantially similar or identical moieties or two or more moieties of the same type. In another embodiment, the anti-MBG antibody fragment comprises two or more moieties of different types, or two or more different types.

The moiety may be human serum albumin (HSA) or a portion thereof (eg, domain III) that binds to the neonatal Fc receptor (FcRn). HSA has a nominal serum half-life of about 19 days due in part to a molecular weight (MW) of about 67 kDa and a high anionic surface.

Covalent or non-covalent binding of the protein to HSA provides additional advantages. For example, HSA masks the protein, so that the protein is less susceptible to proteolytic cleavage and less immunogenic.

The major FcRn binding site is in domain III of HSA. Domain III spans positions 388-585 of the 609-aa full length HSA. In addition, domain I contributes to HSA binding to FcRn, which may partially explain why domain III as a single domain has about 10 times lower affinity for FcRn than full length HSA.

The HSA or FcRn-binding portion thereof may optionally have one or more mutations that provide beneficial properties or effects. In some embodiments, the HSA or FcRn-binding portion thereof has one or more mutations that enhance pH-dependent HSA binding to FcRn or/and increase HSA half-life, such as K573P or/and E505G/V547A.

The HSA or FcRn-binding portion thereof can be attached to any suitable position or site on the anti-MBG antibody fragment. In some embodiments, the HSA or FcRn-binding portion thereof is recombinantly fused or chemically conjugated to the N-terminus or/and C-terminus of the heavy or/and light chain of the anti-MBG antibody fragment, optionally via a linker.

In another embodiment, the extending moiety is or comprises a moiety that non-covalently binds to albumin (HSA). Attachment of the anti-MBG antibody fragment to the albumin binding moiety allows the antibody fragment to enjoy the same advantages as attaching the antibody fragment to the HSA. In addition, non-covalent binding to HSA enables slow and sustained release of antibody fragments from the HSA-antibody fragment complex. In addition, albumin binding moieties enable affinity purification of antibody fragments based on binding to albumin.

In other embodiments, the extension moiety is or comprises an unstructured polypeptide. Attachment of an unstructured polypeptide to a protein increases its MW, hydrodynamic radius/volume and overall size, thereby increasing its half-life, reducing the rate of kidney clearance. Unstructured polypeptides are usually intermolecular interactions or water molecules with a backbone that adopts any chain form in aqueous solution at body temperature, and water molecules that prefer small, hydrophilic amino acids with side chains that are not generally charged under physiological conditions. It has a side chain with a low tendency to be involved in intermolecular interactions other than intermolecular interactions with and. Hydrophobic amino acids may be involved in intramolecular or intermolecular interactions, including non-specific hydrophobic interactions with cell membranes and cellular components. Among the uncharged hydrophilic amino acids, threonine can form β-sheets, cysteine can form disulfide bonds, tyrosine is somewhat larger, phenyl ring can be involved in hydrophobic interactions, and asparagine and glutamine aggregates Can be formed.

The unstructured polypeptide can be attached to any suitable position or site on the anti-MBG antibody fragment. In some embodiments, the unstructured polypeptide is recombinantly fused or chemically conjugated to the N-terminus or/and C-terminus of the heavy or/and light chain of the anti-MBG antibody fragment, optionally via a linker. Likewise, anti-MBG antibody fragments can be attached to any suitable location or site on the unstructured polypeptide. In some embodiments, the anti-MBG antibody fragment is recombinantly fused or chemically conjugated to the N-terminus or C-terminus of the unstructured polypeptide, optionally via a linker.

In a further embodiment, the extending moiety comprises or is a carboxy-terminal peptide (CTP) derived from the β-subunit of human chorionic gonadotropin (hCG). The extended CTP (CTP attached to the protein as an extended moiety) may have the wild-type sequence of hCG-β CTP, or it may have one or more substitutions, additions or deletions, or any combination or all thereof compared to hCG-β CTP. I can have it. CTP can be attached to any suitable location or site on the anti-MBG antibody fragment. In some embodiments, the CTP is recombinantly fused or chemically conjugated to the N-terminus or/and C-terminus of the heavy or/and light chain of the anti-MBG antibody fragment, optionally via a linker. Likewise, anti-MBG antibody fragments can be attached to any suitable location or site on the CTP. In some embodiments, the anti-MBG antibody fragment is recombinantly fused or chemically conjugated to the N-terminus or C-terminus of CTP, optionally through a linker.

In some embodiments, the polypeptide extension moiety is recombinantly fused to the N-terminus or C-terminus of the heavy or light chain of the anti-MBG antibody fragment, optionally via a linker. Recombinant fusion of the polypeptide extension moiety to the antibody fragment facilitates production, inhibits further conjugation and purification steps, and minimizes product heterogeneity. In certain embodiments, the polypeptide extension moiety is recombinantly fused to the anti-MBG antibody fragment via a linker. Linkers/spacers reduce steric hindrance and promote the independent function of antibody fragments and extension moieties. In addition, the use of linkers can enhance the expression of the fusion protein. In some embodiments, the linker contains about 4-30 amino acid residues. In certain embodiments, the linker contains about 6 or 8 amino acid residues to about 20 amino acid residues, or about 6 or 8 amino acid residues to about 15 amino acid residues.

In other embodiments, the extension moiety comprises or is a moiety of 1, 2, 3, 4, 5 or more moieties of a synthetic polymer. Synthetic polymers can be biodegradable or non-biodegradable. Biodegradable polymers useful as extension moieties include, but are not limited to, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). Does not. Non-biodegradable polymers useful as extension moieties include, without limitation, poly(ethylene glycol) (PEG), polyglycerol, poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polyoxazoline and poly( N-vinylpyrrolidone) (PVP).

In some embodiments, the synthetic polymer comprises or is PEG. Conjugation of one or more PEG moieties to a protein increases its half-life by increasing its MW, hydrodynamic radius/volume and overall size, thereby reducing the rate of kidney clearance. Pegylation can increase solubility, reduce aggregation and immunogenicity, and inhibit phagocytosis of proteins. PEG is flexible, non-fillable, non-biodegradable, relatively low immunogenic, and approved by the US Food and Drug Administration as GRAS (generally recognized as safe). One or more PEG moieties may independently be linear or branched. In addition, one or more PEG moieties may be independently terminated at a hydroxyl group, a methoxy group (mPEG) or other capped group.

In some embodiments, the individual mass (e.g., average molecular weight), or total mass, of one or more synthetic polymer moieties is about 10-50, 10-20, 20-30, 30-40 or 40-50 kDa, or about 10, 20, 30, 40 or 50 kDa. The individual mass (e.g., average MW), or total mass, of one or more synthetic polymer moieties is about 50-100, 50-60, 60-70, 70-80, 80-90 or 90-100 kDa, or about It may be greater than about 50 kDa, such as 60, 70, 80, 90 or 100 kDa. In addition, the mass (eg, average MW) of individual synthetic polymer moieties may be less than about 10 kDa, such as about 1-5 or 5-10 kDa, or about 5 kDa. In certain embodiments, the individual mass (eg, average MW), or total mass, of one or more synthetic polymer (eg, PEG) moieties is about 20 or 40 kDa. The half-life of the modified protein can be adjusted according to the length of the synthetic polymer, and generally longer polymers impart a longer half-life.

One or more synthetic polymeric moieties can be coupled to any suitable position(s) or site(s) on the anti-MBG antibody fragment. In some embodiments, the one or more synthetic polymeric moieties are N-terminus, C-terminus, and/or one or more side chains of the heavy or/and light chain of the anti-MBG antibody fragment, optionally via a linker [e.g., an accessible lysine side chain amino group. Or cysteine thiol group(s)].

In some embodiments, the anti-MBG antibody fragment comprising one or more extension moieties is sdAb, scFv, Fv or Fab. In certain embodiments, the anti-MBG antibody fragment is Fab 2A-F8.

As a non-limiting example, a non-human (eg, murine), humanized or human MBG-binding V _H or V _L domain can be recombinantly fused to a polypeptide extension moiety. Such anti-MBG V _H -or V _{L -extension moiety fusion proteins can be used alone or the corresponding V L} or V _H domains which can be optionally fused to the same extension moiety, a different extension moiety or a non-extending polypeptide. (Eg, via a disulfide bond or a linker) (eg, _{a V H} -extension moiety linked to a V _{L-extension moiety).}

IV. Pharmaceutical composition

Further aspects of the present disclosure include chimeric, humanized or human anti-MBG antibodies or fragments thereof described herein, or pharmaceutically acceptable salts, solvates or hydrates thereof, and one or more pharmaceutically acceptable excipients or It relates to a pharmaceutical composition comprising a carrier. This composition may optionally contain additional therapeutic agents. In general, the pharmaceutical composition contains a therapeutically effective amount of an anti-MBG antibody or fragment thereof, one or more pharmaceutically acceptable excipients or carriers and optionally a therapeutically effective amount of an additional therapeutic agent, and is administered to a subject for therapeutic use. Formulated for. In addition, anti-MBG antibodies or fragments thereof can be administered to a subject for diagnostic use such as imaging.

Pharmaceutical compositions are generally of current good manufacturing practice as recommended or required by, for example, the International Conference on Federal Food, Drug and Cosmetic Act §501 (a) (2) (B) and Harmonisation Q7 guidelines. Manufactured in accordance with management standards (GMP).

Pharmaceutical compositions/formulations can be prepared in sterile form. For example, pharmaceutical compositions/formulations for parenteral administration by injection or infusion are generally sterilized. The sterilized pharmaceutical compositions/formulations are synthesized or prepared according to pharmaceutical grade sterilization standards known to those of skill in the art as published or required in U.S. Pharmacopoeia chapters 797, 1072 and 1211 and 21 Code of Federal Regulations 211.

Pharmaceutically acceptable excipients and carriers include pharmaceutically acceptable substances, materials and vehicles. Non-limiting examples of types of excipients include liquid and solid fillers, diluents, binders, lubricants, lubricants, surfactants, dispersants, disintegrants, emulsifiers, wetting agents, suspending agents, thickeners, solvents, isotonic agents, buffers, pH adjusters, Absorption delaying agents, stabilizers, antioxidants, preservatives, antimicrobial agents, antibacterial agents, antifungal agents, chelating agents, adjuvants, sweetening agents, flavoring agents, coloring agents, encapsulating materials and coating materials. The use of such excipients in pharmaceutical formulations is known in the art. For example, conventional carriers and carriers include, without limitation, oils (eg, vegetable oils such as olive oil and sesame oil), aqueous solvents {eg, saline, buffered saline (eg, phosphate buffered saline [PBS]). ) And isotonic solutions (eg Ringer's solution)}, and organic solvents (eg dimethyl sulfoxide [DMSO] and alcohols [eg ethanol, glycerol and propylene glycol]). Except where any conventional excipients or carriers are incompatible with anti-MBG antibodies or fragments thereof, the present disclosure includes the use of conventional excipients and carriers in formulations containing anti-MBG antibodies or fragments thereof. do. See, eg, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (Philadelphia, Pennsylvania) (2005); Handbook of Pharmaceutical Excipients, 5th Ed., Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association (2005); Handbook of Pharmaceutical Additives, 3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); and Pharmaceutical Pre-formulation and Formulation, Gibson, Ed., CRC Press (Boca Raton, Florida) (2004).

Proper formulation may depend on a variety of factors, such as the route of administration chosen. Potential routes of administration of pharmaceutical compositions comprising anti-MBG antibodies or fragments thereof are, without limitation, oral, parenteral (intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intramedullary, intrathecal and Topical), intranasal, and topical (skin/epithelial, transdermal, mucosal, transmucosal, intranasal [eg, by nasal spray or instillation], intraocular [eg, by eye drops], lungs [eg Examples include oral or nasal inhalation], buccal, sublingual, rectal [eg, by suppository], and vaginal [eg, by suppository]). Topical formulations can be designed to produce a local or systemic therapeutic effect. In certain embodiments, the anti-MBG antibody or fragment thereof is by injection (e.g., as a bolus) parenterally (e.g., intravenous, subcutaneous, intramuscular or intraperitoneal) or by infusion over a period of time. Is administered.

Excipients and carriers that can be used to prepare parenteral formulations include, without limitation, solvents (eg, water, saline, physiological saline, buffered saline [eg, phosphate buffered saline], balanced salt solutions [eg, Ringer BSS). ] And aqueous dextrose solutions), isotonic/etc. osmotic agents (eg salts [eg NaCl, KCl and CaCl ₂ ] and sugars [eg sucrose]), buffers and pH adjusters (eg For example, sodium dihydrogen phosphate [sodium monobasic phosphate]/disodium hydrogen phosphate [sodium dibasic phosphate], citric acid/sodium citrate and L-histidine/L-histidine HCl), and emulsifiers (e.g., polysorbate Polysorbates (eg polysorbates 20 and 80) and nonionic surfactants such as poloxamers (eg poloxamer 188). Protein formulation and delivery systems are discussed, for example, in AJ Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd Ed., CRC Press (Boca Raton, Florida) (2015).

The excipient may optionally include one or more substances, or any combination or all thereof, that increase protein stability, increase protein solubility, inhibit protein aggregation or reduce solution viscosity. Examples of such substances include, without limitation, hydrophilic amino acids (e.g. arginine and histidine), polyols (e.g. myo -inositol, mannitol and sorbitol), sugars (e.g., glucose (D-glucose [Including dextrose]), lactose, sucrose and trehalose}, osmolytes (eg, trehalose, taurine, amino acids [eg, glycine, sarcosine, alanine, proline, serine, β-alanine and γ-amino butyric acid], and betaines [eg trimethylglycine and trimethylamine N-oxide]), and nonionic surfactants {eg alkyl polyglycosides, ProTek® alkylsaccharides (eg, Monosaccharides [e.g. glucose] or disaccharides [e.g. maltose or sucrose]) bound to long chain fatty acids or corresponding long chain alcohols and polypropylene glycol/polyethylene glycol block copolymers (e.g. poloxamer [e.g. For example, Pluronic ^™ F-68], and Genapol ^® PF-10 and variants thereof)}. Because these substances increase protein solubility, they can be used to increase protein concentration in formulations. The higher protein concentration in the formulation is particularly advantageous for subcutaneous administration with limited volumes of bolus administration (eg, up to about 1.5 mL). In addition, these materials can be used to stabilize proteins in the process of manufacturing, storage and reconstitution of lyophilized proteins.

For parenteral (e.g., intravenous, subcutaneous or intramuscular) administration, a sterile solution or suspension of an anti-MBG antibody or fragment thereof in an aqueous solvent containing one or more excipients may be prepared in advance, for example , May be provided in a pre-filled syringe. Alternatively, the anti-MBG antibody or fragment thereof may be dissolved or suspended in an aqueous solvent, which may optionally contain one or more excipients, prior to lyophilization (lyophilization). Immediately prior to parenteral administration, the lyophilized anti-MBG antibody or fragment thereof stored in a suitable container (eg, vial) can be reconstituted with sterile water, which may optionally contain, for example, one or more excipients. When the anti-MBG antibody or fragment thereof is administered by infusion (e.g., intravenous), a solution or suspension of the reconstituted anti-MBG antibody or fragment thereof is, for example, sterile saline (e.g., about 0.9 % NaCl) and can be diluted.

Excipients that enhance the penetration of smaller proteins into transmucosal oils include, without limitation, cyclodextrins, alkyl saccharides (eg, alkyl glycosides and alkyl maltosides [eg, tetradecyl maltoside]) and bile acids. (E.g., cholic acid, glycocholic acid, tarocholic acid, deoxycholic acid, glycodeoxycholic acid, kenodeoxy cholic acid, and dehydrocholic acid).

Excipients that enhance transepithelial or transdermal penetration of smaller proteins are, without limitation, chemical penetration enhancers (CPE containing fatty acids [eg, oleic acid]), cell penetrating peptides {CPP including arginine-rich CPP [eg, Poly arginine such as R ₆ -R _{11 (eg R 6} and R ₉ ) and TAT related CPP such as TAT (49-57)] and amphiphilic CPP [eg Pep-1 and penetratin) ]}, and skin-penetrating peptides (SPPs such as skin-penetrating and cell-entry [SPACE] peptides). Transdermal penetration of smaller proteins can be further enhanced using physical enhancement techniques such as iontophoresis, cavitational or non-cavitation ultrasound, electroporation, thermal ablation, radio frequency, microdermabrasion, microneedle or jet implantation. . US 2007/0269379 provides an extensive list of CPEs. F. Milletti, Drug Discov. Today , 17 :850-860 (2012) is a review of CPPs. R. Ruan et al ., Ther. Deliv ., 7 :89-100 (2016) discuss CPP and SPP for transdermal delivery of macromolecules, and M. Prausnitz and R. Langer, Nat. Biotechnol ., 26 :1261-1268 (2008) discusses various transdermal drug-delivery methods.

In some embodiments, the anti-MBG antibody or fragment thereof is delivered from a sustained release composition. The term “sustained-release composition” as used herein includes sustained release, long-lasting release, extended release, sustained and controlled release compositions, systems and devices. Protein delivery systems are discussed in Banga (above), for example. Sustained release compositions are capable of delivering a therapeutically effective amount of an anti-MBG antibody or fragment thereof over a long period of time. In some embodiments, the sustained-release composition contains the anti-MBG antibody or fragment thereof over a period of at least about 3 days, 1 week, 2 weeks, 3 weeks, 1 month (4 weeks), 6 weeks, 2 months, 3 months or longer. Deliver. The sustained composition may be administered parenterally (eg, intravenously, subcutaneously or intramuscularly), for example.

The sustained release composition of the protein may be in the form of, for example, a particulate system, a lipid or greasy composition, or an implant. Particulate systems include, without limitation, nanoparticles, nanospheres, nanocapsules, microparticles, microspheres and microcapsules. Nanoparticulate systems generally have a diameter or equivalent dimension less than about 1 μm. In certain embodiments, the nanoparticles, nanospheres, or nanocapsules have a diameter or equivalent dimension of about 500, 400, or 300 nm or less, or about 200, 150, or 100 nm or less. In some embodiments, the microparticles, microspheres, or microcapsules have a diameter or equivalent dimension of about 1-200, 100-200 or 50-150 μm, or about 1-100, 1-50 or 50-100 μm. Nano- or microcapsules generally contain the therapeutic agent in a central core, but the therapeutic agent is generally dispersed throughout nano- or microparticles or spheres. In certain embodiments, the nanoparticulate system is administered intravenously, while the microparticulate system is administered subcutaneously or intramuscularly.

In some embodiments, the sustained release particulate system or implant is made of biodegradable polymers and/or hydrogels. In certain embodiments, the biodegradable polymer is lactic acid or/and glycolic acid (e.g., poly(L-lactide-co-glycolide) or poly(L-lactic acid-co-D,L-2-hydroxyoctanoic acid) ), such as L-lactic acid-based copolymer]. Non-limiting examples of polymers from which the hydrogel can be constituted include polyvinyl alcohol, acrylate polymer (e.g., sodium polyacrylate), and a relatively large number of hydrophilic groups (e.g., hydroxyl or/and Carboxylate groups) and other single polymers and copolymers. The biodegradable polymer of the particulate system or implant can be selected such that the polymer is substantially completely degraded at the time the treatment period is expected to end, and by-products of polymer degradation, such as the polymer, become biocompatible.

Alternatively, the sustained release composition of the protein can be composed of a non-biodegradable polymer. Examples of non-biodegradable polymers include, without limitation, poloxamers (eg, poloxamer 407). The sustained release composition of the protein may be composed of other natural or synthetic substances or materials such as hydroxyapatite.

Sustained release lipids or greasy compositions of proteins can be in the form of emulsions of, for example, liposomes, micelles (e.g., those composed of biodegradable natural or/and synthetic polymers such as lactosomes) and oils.

Sustained release compositions may be formulated or designed as depots that can be injected or implanted, for example subcutaneously or intramuscularly. The reservoir may be in the form of, for example, a polymeric particulate system, a polymeric implant or a lipid or oily composition. Reservoir formulations include proteins and, for example, a biodegradable polymer [eg, poly(lactide-co-glycolide)] or a semi-biodegradable polymer (eg, a block of lactic acid and PEG). Copolymers) can be included in a biocompatible solvent system, regardless of whether such mixtures form particulate systems or implants.

Pharmaceutical compositions may be presented in unit dosage form as a single dose, where all active and inactive ingredients are combined in a suitable system and the ingredients need not be mixed to form the composition to be administered. The unit dosage form generally contains an effective amount of the therapeutic agent. A representative example of a unit dosage form is a disposable pen comprising a prefilled syringe, a needle and a needle cover for parenteral (eg, intravenous, subcutaneous or intramuscular) injection of a therapeutic agent.

Alternatively, the pharmaceutical composition is a composition to be administered in combination, wherein the therapeutic agent, excipient and carrier (e.g., solvent) are provided in two or more separate containers (e.g., ampoules, vials, tubes, bottles or syringes). It may be provided as a kit that needs to be combined to form. The kit may include instructions for storing, preparing and administering the composition (eg, a solution for intravenous or subcutaneous injection).

Kits may contain all the active and inactive ingredients in unit dosage form or in two or more separate containers, and may contain instructions for administering or using the pharmaceutical composition to treat a medical condition.

In some embodiments, the kit comprises instructions for administering or using an anti-MBG antibody or fragment thereof or a pharmaceutical composition comprising the same, and an anti-MBG antibody or fragment thereof or a pharmaceutical composition comprising the same to treat an MBG-related disease. Includes.

V. Therapeutic use of anti-MBG antibodies and fragments thereof

The anti-MBG antibodies described above can be administered to a subject having an MBG-related disease or syndrome. When the subject is a human, the anti-MBG antibody may be a chimeric mouse-human antibody, a humanized antibody, or a human antibody. Such chimeric, humanized and human antibodies are detailed. MBG-related diseases and syndromes include, for example, acute respiratory distress syndrome, traumatic brain injury, high blood pressure (e.g., essential hypertension), hypertension-related diseases (e.g., Pre-eclampsia) and fibrosis-related diseases (eg, non-alcoholic steatohepatitis, diabetic kidney disease). MBG-related diseases also include severe patients with a background of sepsis, trauma, complex severe pneumonia and burns.

Increased levels of marinobufagenin (MBG) are associated with subjects with acute respiratory distress syndrome. Acute respiratory distress syndrome (ARDS) is a life-threatening disease with a prevalence of 64 cases per 100,000 people per year and a mortality rate of 40 to 52%. MBG-related effects of acute respiratory distress syndrome can be solved by administering anti-MBG antibodies to the subject. Anti-MBG antibodies remove MBG from the subject, thus lowering increased levels of MBG. Reducing the MBG level should reduce the detrimental effects of increased MBG.

Elevated levels of marinobufagerin (MBG) are associated with subjects suffering from traumatic brain injury (eg, concussion). MBG not only causes inflammation, but also maintains the inflammatory response. Traumatic brain injury is associated with increased permeability of the blood brain barrier, and MBG can cross the BBB and enter the central nervous system. When anti-MBG antibodies are administered systemically or directly to the CNS, they can either remove MBG from the system (and lower CNS MBG by allowing more CNS MBG to enter the blood), or lower MBG in the CNS by antibody-immobilized removal. have. Reducing MBG levels can reduce CNS inflammation in a subject and can reduce other MBG-related effects in the CNS.

Essential hypertension, also known as primary or idiopathic hypertension, affects about 90-95% of hypertensive patients. Essential hypertension occurs primarily due to vasoconstriction or volume expansion (Laragh 1982). Volume expansion is an increase in fluid in the blood due to increased sodium and water retention. Excessive volume expansion can be caused by, for example, high dietary NaCl intake or lower sodium handling in patients with disorders such as congestive heart failure (CHF), renal failure or liver failure. Marinobufagenin (MBG) is a cardiac enhancing steroid (CTS) with effects similar to digitalis. MBG is an important regulator of blood pressure and is associated with the etiology of hypertension and hypertension-related disorders. MBG is a powerful vasoconstrictor (Bagrov, EJP 1995). In addition, volume expansion induces MBG synthesis and secretion, including volume expansion related conditions such as essential hypertension, primary aldosteronism, preeclampsia (PE), CHF and chronic renal failure (Bagrov 2009). MBG is a factor that causes preeclampsia through, for example, induction of vasoconstriction and vascular fibrosis. Administration of MBG to normal pregnant rats causes an increase in blood pressure similar to that observed in pregnant rats drinking saline and injected with desoxycorticosterone acetate (DOCA), the aldosterone precursor, a volume expansion rat model of PE (Vu 2005). . Plasma MBG levels are 5 times higher in PE patients than in normal blood pressure pregnant women, and plasma MBG levels correlate with PE severity (Lopatin 1999 and Agunanne 2011). In addition, plasma MBG levels increase 7-fold in essential hypertension or CHF patients and 70-fold or more in chronic renal failure patients (Puschett, AJKD 2010).

Increased plasma MBG levels include pre-eclampsia (Graves 1987), congestive heart disorder (Schwinger 1999), myocardial ischemia/infarction (Bagrov 1991), diabetes (including type 1 and type 2) (Bagrov 2005), and obstructive sleep. It is associated with a variety of diseases characterized by ^{reduced Na +} /K ⁺ -ATPase (NKA) pumping activity, including apnea (Bagrov, Hypertension 1995 and Paci 2000).

MBG inhibits the proliferation, migration and invasion of cytotrophic sublayer (CTB) cells, which are important for normal placental development (LaMarca 2006 and Uddin, Placenta 2008). In about 7% of all births, PE is characterized by the inability of CTB cells to invade the uterus and its vascular system, particularly the spiral arteries, which should be invaded by the intravascular CTB cells. This reduces blood flow to the fetus through the placenta, which can lead to intrauterine growth restriction (IUGR). In addition, MBG binding to NKA induces p38 mediated apoptosis and stress signaling in the extrachorionic CTB cell line (Ehrig 2015). In addition, MBG has apoptosis mediated by MAPKs p38 and c-Jun NH ₂ -terminal kinase (Jnk), and p38 mediated production and secretion of IL-6 in extrachorionic CTB cell lines (cell proliferation, cell differentiation, apoptosis and Mediates inflammation) (Uddin, JBC 2008).

In addition, MBG induces apoptosis of endothelial cells, which increases the vascular permeability/leakage properties of PE, and causes endothelial dysfunction by destroying tight junctions (Uddin, AJN and AJPRICP 2009). In addition, vascular inflammation as well as endothelial dysfunction contribute to increased peripheral resistance and vascular damage in hypertension (Versari 2009 and Marchesi 2008).

MBG binding to NKA stimulates EGFR-mediated cellular signaling, leading to oxidative stress and fibrosis (Bagrov 2008). MBG induces fibrosis in kidney and cardiovascular tissues, including cardiac fibrosis in uremic cardiomyopathy (Kennedy 2006 and Elkareh 2007 and 2009). Fibrosis is the formation of excessive fibrous connective tissue in an organ or tissue in the process of recovery or responsiveness, usually in response to injury or inflammation. Fibrosis involves stimulated fibroblasts that underlie connective tissue, including collagen and glycosaminoglycans. Along with elastin, collagen is a major fibrous sclerotic protein in connective tissue. The transcription factor Fli-1 is a negative regulator (inhibitor) of procollagen I and III transcription and inhibits TGF-β-induced collagen synthesis (Kubo 2003 and Czuwara-Ladykowska 2001). Binding of low concentration MBG to NKA on fibroblasts activates PLC and PKC-δ as described above. Activation of PKC-δ induces translocation to the nucleus, phosphorylating Fli-1, making Fli-1 susceptible to proteolysis. Protein degradation of phosphorylated Fli-1 results in increased expression of procollagen I and III, collagen synthesis, and thus fibrosis (Elkareh 2009). Type I collagen is the most abundant collagen in the human body, forms collagen fibers, and is present in scar tissue. Type III collagen is often associated with type I collagen and is present in expandable connective tissues such as the lungs, skin and vascular system. Fli-1 participates in the pathogenesis of various fibrotic diseases, including focal and systemic scleroderma, and Fli-1 deficiency promotes collagen production in fibrotic skin and vascular lesions (Kubo 2003, and Asano AJP and JDS 2010).

MBG induces vascular fibrosis in PE (Nikitina 2011), and arterial stiffness is characterized by preeclampsia and chronic hypertensive pregnancy (Tihtonen 2006 and Kaihura 2009). Umbilical arteries of PE patients showed decreased Fli-1 expression, increased expression of procollagen I and III, impaired response to the vasodilating effect of sodium nitroprusside after endothelin-1-induced vasoconstriction, and for 24 hours. In vitro treatment of normal umbilical artery explantation with 1 and 10 nM MBG mimics the same function of PE (Nikitina 2011).

MBG induces epithelial-mesenchymal metastasis (EMT) in renal epithelial cells, in part, causing periglomerular and perivascular fibrosis of the renal cortex and the formation of fibrous scars at the cortical nodular junction of the kidney (Fedorova 2009). Sub-nanomolar concentrations of MBG strongly stimulate collagen synthesis by normal kidney and cardiac fibroblasts (Elkareh 2009 and 2007). NKA binds to Src kinase and functions as a signal transduction complex in response to CTS-like MBG binding to NKA in kidney and cardiovascular cells (Tian 2006). The activation of Src is required for the induction of collagen synthesis by fibroblasts (Elkareh 2007).

Fibrosis impairs the structure and function of the affected organ (eg, heart or kidney) or tissue (eg, the vascular or renal tubular system). With respect to DN, MBG binds to NKA in renal fibroblasts and renal epithelial cells (including cells of the renal tubular system), and at low concentrations, MBG does not inhibit NKA pumping and NKA/Src-initiated leading to collagen production and fibrosis. Induces the signaling cascade. _{MBG preferentially binds to the α 1} isoform of NKA, the major isoform of the kidney, including renal fibroblasts and renal epithelial cells (including cells of the renal tubular system) (Bagrov 2009). MBG activation of the NKA/Src signal transduction complex is renal fibroblasts and fibrosis by promoting proteolysis of transcription factor Fli-1, a factor that inhibits the expression of procollagen I and III, by PKC-β phosphorylation of Fli-1 as described above. By inducing collagen production. In addition, MBG induces EMT, causing fibrotic changes in the epithelial cells of the renal tubular system, including the proximal tubule (Fedorova 2009).

Fibrosis is associated with a variety of diseases. Examples of diseases in which fibrosis occurs are, without limitation, preeclampsia, cardiomyopathy (e.g., diabetic cardiomyopathy and uremic cardiomyopathy), cardiac fibrosis, myocardial fibrosis, collagen. Vascular diseases (e.g., atherosclerosis and vascular fibrosis), atherosclerosis, chronic heart failure, diabetic kidney disease, glomerulosclerosis, renal fibrosis, tubulointerstitial fibrosis ), chronic kidney disease (e.g., chronic kidney failure), liver fibrosis, cirrhosis, nonalcoholic fatty liver disease (e.g., nonalcoholic fatty hepatitis), alcoholic liver disease (e.g., alcoholic fatty hepatitis), Chronic liver disease, liver failure (e.g., cirrhosis and chronic liver failure), pulmonary fibrosis, renal systemic fibrosis (which may affect the skin and internal organs such as the heart, liver and lungs), and scleroderma (localized Scleroderma and systemic scleroderma/systemic sclerosis (which may affect skin, blood vessels, muscles and internal organs such as the heart, kidneys, lungs and gastrointestinal tract).

In addition, hypertension is characterized by a variety of diseases (eg, clinical features or complications) or is a major risk factor (eg, cause or contribution). Non-limiting examples of diseases associated with hypertension include preeclampsia, eclampsia, edema (e.g., systemic edema, peripheral edema, pulmonary edema and cerebral edema), cardiovascular disease (e.g. For example, cardiomyopathy [eg, diabetic cardiomyopathy and uremic cardiomyopathy], myocardial fibrosis and collagen vascular disease [eg, arterial stiffness]), arteriosclerosis (eg, atherosclerosis and arteriosclerosis) , Heart failure (e.g., congestive heart failure, left heart failure and right heart failure), coronary artery disease (e.g. myocardial ischemia/infarction), aortic dissection, peripheral vascular disease (e.g., peripheral arterial disease ) And stroke}, kidney disease (e.g., hypertensive kidney disease, diabetic kidney disease, glomerulonephritis, renal ischemia, nephrotic syndrome), renal failure (e.g., acute kidney injury and chronic kidney Disease], hematuria, proteinuria and uremia), liver failure (eg, cirrhosis), brain disease (eg, hypertensive encephalopathy, cerebral hemorrhage), intracranial hemorrhage, intracranial hemorrhage Hypertension, stroke [eg, ischemic stroke and hemorrhagic stroke], and cerebral infarction), eye diseases (eg, retinopathy) [eg, hypertensive retinopathy], eye Vascular damage, optic nerve papilledema, retinal hemorrhage and vision loss), fibrinoid necrosis (e.g. arterioles), metabolic syndrome, diabetes (e.g., type 2) and sleep Apnea (eg obstructive sleep apnea).

The chimeric, humanized and human anti-MBG antibodies and fragments thereof described herein can be used to treat any MBG-related disease. In certain embodiments, the antibody or fragment thereof is H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8. In some embodiments, the disease is associated with increased levels of MBG, such as blood, urine or cerebrospinal fluid (CSF). Non-limiting examples of MBG-related diseases include hypertension (eg, essential [primary or idiopathic] hypertension, secondary hypertension [eg, renal vascular hypertension, portal hypertension or pulmonary hypertension], volume expansion-related hypertension, resistant hypertension. And hypertension emergency [malignant] hypertension]), pregnancy hypertension disease (eg, chronic hypertension, preeclampsia, eclampsia and gestational hypertension), edema (eg, systemic edema and peripheral edema), cardiovascular disease (eg, Hypertensive heart disease, fibrotic disease (eg, cardiomyopathy [eg, diabetic cardiomyopathy and uremic cardiomyopathy], heart fibrosis, myocardial fibrosis [eg, endocardial fibrosis] and collagen-vascular disease [eg For example, arterial stiffness and vascular fibrosis]), atherosclerosis (eg, atherosclerosis and atherosclerosis), heart failure (eg, acute heart failure, chronic heart failure, congestive heart failure, left heart failure/left ventricular dysfunction and Right heart failure), coronary artery disease (eg, angina and myocardial ischemia/infarction), aortic dissection, peripheral vascular disease (eg, peripheral artery disease) and stroke}, kidney/kidney disease {eg, hypertensive Kidney disease, diabetic kidney disease, glomerulonephritis, glomerulosclerosis, renal fibrosis, tubular interstitial fibrosis, renal ischemia, nephrotic syndrome, renal failure (e.g., acute kidney injury, acute chronic renal failure and chronic kidney disease) [for example, Chronic renal failure and end-stage kidney disease]), hematuria, proteinuria and uremia}, liver disease {eg, liver fibrosis, cirrhosis, non-alcoholic fatty liver disease (eg, non-alcoholic steatohepatitis), alcoholic liver disease ( For example, alcoholic steatohepatitis), chronic liver disease and liver failure (eg, cirrhosis and chronic liver failure)}, lung disease (eg, pulmonary edema and pulmonary fibrosis [eg, idiopathic pulmonary fibrosis]), brain disease (For example, hypertensive encephalopathy, cerebral hemorrhage, intracranial hemorrhage, cerebral edema, intracranial hypertension, brain swelling, stroke [for example, stroke and brain Infarction], and cerebral infarction), eye diseases (eg, retinopathy [eg, hypertensive retinopathy], blood vessel damage of the eye, papilloedema [papillary edema], retinal hemorrhage, and vision impairment, and Loss), skin diseases (e.g., nephrogenic systemic fibrosis [may also affect internal organs such as heart, liver and lungs) and scleroderma (localized and systemic scleroderma/systemic sclerosis [vascular, muscle And internal organs such as the heart, kidneys, lungs and gastrointestinal tract)), fibrin necrosis (eg, arterioles), metabolic syndrome, diabetes mellitus (including types 1 and 2), and sleep apnea (E.g. obstructive sleep apnea).

In some embodiments, chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) are used to treat hypertensive diseases. In certain embodiments, the high blood pressure comprises or is high arterial blood pressure. In some embodiments, the hypertensive disorder is hypertension (e.g., essential hypertension, volume expansion-related hypertension or resistant hypertension), pregnancy hypertension disorder (e.g., chronic hypertension or preeclampsia), or other hypertension-related disorders (e.g. , Cardiovascular disease (eg, hypertensive heart disease, heart failure [eg, congestive heart failure] or coronary artery disease [eg, myocardial ischemia/infarction]) or kidney disorder (eg, hypertensive kidney disease, Diabetic kidney disease or renal failure [eg, chronic kidney disease])}. In certain embodiments, the hypertensive disease is essential hypertension, pre-eclampsia, congestive heart failure, or chronic kidney disease.

In other embodiments, chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) are used to treat fibrotic diseases. In some embodiments, the fibrotic disease is preeclampsia, cardiomyopathy (e.g., diabetic cardiomyopathy or uremic cardiomyopathy), cardiac fibrosis, myocardial fibrosis, collagen-vascular disease (e.g., arterial stiffness or vascular fibrosis), Atherosclerosis, chronic heart failure, diabetic kidney disease, kidney fibrosis, chronic kidney disease (e.g., chronic kidney failure), liver fibrosis, cirrhosis, nonalcoholic steatohepatitis, chronic liver disease, liver failure (e.g., chronic liver failure) or Scleroderma (eg, local scleroderma or systemic scleroderma/systemic sclerosis). In certain embodiments, the fibrotic disease is uremic cardiomyopathy, myocardial fibrosis, diabetic kidney disease, renal fibrosis or cirrhosis.

Chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) can be administered to a subject via any suitable route. . Potential routes of administration of the antibody or fragment thereof include, without limitation, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial and intraperitoneal), and intranasal. In some embodiments, the antibody or fragment thereof is administered parenterally (eg, intravenously, subcutaneously, intramuscularly or intraperitoneally) by injection or infusion (eg, using an osmotic pump). In addition, antibodies or fragments thereof may be administered by different routes, in part depending on the type of disease. For example, the antibody or fragment thereof can be administered to treat a pulmonary disorder, for example using a metered dose inhaler, dry powder inhaler or nebulizer, for example by oral inhalation. As another example, the antibody or fragment thereof can be administered to treat a brain disease, for example by intramedullary, intrathecal, intracranial, intracranial or intracranial injection. As another example, the antibody or fragment thereof can be administered to treat an ocular disease, for example by intravitreal, subconjunctival, subretinal or sub-Tenon's injection or implantation. Alternatively, the antibody or fragment thereof can be administered topically as an eye drop to treat an ocular disease, wherein penetration of the antibody or fragment thereof into the posterior segment or/and anterior segment of the eye can result in an arginine-rich CPP [eg, Polyarginine such as R _{6 -R 11} (e.g. R ₆ or R ₉ ) or TAT related CPP such as TAT (49-57)] or amphiphilic CPP (e.g. Pep-1 or penetatin ( penetratin)). As a further example, an antibody or fragment thereof can be administered transdermally, for example to treat a skin condition, wherein the penetration of the antibody or fragment thereof into and through the skin is a chemical penetration enhancer (CPE) (e.g. , Fatty acids such as oleic acid), CPP (eg, arginine-rich CPP such as polyarginine or TAT-related CPP) or skin penetrating peptide (SPP) (eg, skin penetrating and cell entry [SPACE] peptide), or By co-administration of any combination or all of these, and/or using physical enhancement techniques such as iontophoresis, cavitation or non-cavitation ultrasound, electroporation, heat removal, radio frequency, microdermabrasion, microneedle or jet implantation. Can be improved.

The therapeutically effective amount and frequency of administration and duration of administration of chimeric, humanized or human anti-MBG antibodies or fragments thereof for the treatment of MBG-related diseases are determined by the nature and severity of the disease, efficacy of the antibody or fragment thereof, mode of administration, age, body weight of the subject. , General health, sex and diet, and may vary depending on a variety of factors including the subject's response to treatment, and may be determined by the treating physician. In some embodiments, a therapeutically effective amount of an antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) for treatment of an MBG-related disease is about 1, 5 Or 10 mg to about 200 mg, about 1, 5 or 10 mg to about 150 mg, about 1, 5 or 10 mg to about 100 mg, or about 1, 5 or 10 mg to about 50 mg, or by the treating physician As deemed appropriate, it may be administered in single or divided doses. In another embodiment, the therapeutically effective amount of the antibody or fragment thereof is about 1-5 mg, 5-10 mg, 10-20 mg, 20-30 mg, 30-40 mg, 40-50 mg, 50-100 mg, 100 -150 mg or 150-200 mg. In some embodiments, the therapeutically effective amount of the antibody or fragment thereof is about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 mg. In certain embodiments, the therapeutically effective amount of the antibody or fragment thereof is about 1-5 mg, 5-10 mg or 10-50 mg. In a further embodiment, a therapeutically effective amount of an antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) for treatment of an MBG-related disease is about 0.01-0.1. mg/kg, 0.1-0.5 mg/kg, 0.5-1 mg/kg, 1-2 mg/kg or 2-3 mg/kg body weight, or as deemed appropriate by the treating physician. In certain embodiments, the therapeutically effective amount of the antibody or fragment thereof is about 0.01-0.1 mg/kg, 0.1-0.5 mg/kg or 0.5-1 mg/kg body weight.

Chimeric, humanized or human anti-MBG antibodies or fragments thereof can be administered at any suitable cycle to treat MBG-related diseases. In some embodiments, the antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) is once a day, once every two days, once every three days, Twice a week, once a week, once every two weeks, once every three weeks, once a month, once every six weeks, once every two months or once every three months, or deemed appropriate by the treating physician If it becomes, it is administered. In certain embodiments, the antibody or fragment thereof is administered once a week or once every two weeks.

Likewise, chimeric, humanized or human anti-MBG antibodies or fragments thereof can be administered for any suitable time period or in any suitable total dose to treat MBG related disorders. In some embodiments, the antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237, or 2A-F8) is at least about 1 week, 2 weeks, 1 month (4 weeks), 6 It is administered for weeks, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or more, or when the treating physician deems appropriate. In some embodiments, the MBG-related disease is a chronic condition. The chronic condition can exist for at least about 6 weeks or 2 months, for example. In certain embodiments, the antibody or fragment thereof is administered over a period of at least about 6 weeks, 2 months, 3 months or 6 months. In a further embodiment, 1, 2, 3, 4, 5 or 6 doses of the antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) are treated as a whole. Is administered for therapy. In certain embodiments, 1, 2 or 3 doses of the antibody or fragment thereof are administered for the entire treatment regimen.

Chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) are administered in an irregular manner to treat MBG-related diseases. It could be. For example, the antibody or fragment thereof may be administered irregularly at least 1, 2, 3, 4, 5 times over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months or 3 months. In addition, anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) can be used as needed (when necessary) for the treatment of MBG-related disorders. Can be consumed. For example, the antibody or fragment thereof may be administered 1, 2, 3, 4, 5 or more times, whether in a regular or irregular manner, for the treatment of hypertension until blood pressure is lowered to a certain level. When blood pressure is reduced to a certain level, administration of the antibody or fragment thereof can be selectively stopped. When blood pressure reaches or exceeds a certain level, administration of the antibody or fragment thereof, whether in a regular or irregular manner, can be resumed. The appropriate dosage, frequency of administration and duration of treatment for the antibody or fragment thereof can be determined by the treating physician.

In order to more quickly establish therapeutic levels of chimeric, humanized or human anti-MBG antibodies or fragments thereof, at least one loading dose of the antibody or fragment thereof may be administered prior to the maintenance dose. In some embodiments, after a loading dose of the antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) is administered, (i) one or more additional loading doses and Thereafter, one or more therapeutically effective maintenance doses, or (ii) one or more therapeutically effective maintenance doses, are administered without an additional loading dose deemed appropriate by the treating physician. The loading dose of the drug is generally larger than the subsequent maintenance dose (e.g., about 1.5, 2, 3, 4 or 5 times greater) and is designed to set the therapeutic level of the drug faster. The one or more therapeutically effective maintenance doses can be any of the therapeutically effective amounts described herein. In certain embodiments, the loading dose is about two or three times greater than the maintenance dose. In some embodiments, the loading dose of the antibody or fragment thereof is administered per day, and the maintenance dose of the antibody or fragment thereof is administered, for example, once weekly during the treatment period or once every two weeks thereafter. In certain embodiments, the antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) is about 2-10 mg, 10-20 mg or 20- 100 mg, or at a loading dose of about 3-15 mg, 15-30 mg or 30-150 mg, followed by once or twice weekly during the treatment period (e.g., for at least about 2, 3 or 6 months) A maintenance dose of about 1-5 mg, 5-10 mg or 10-50 mg once a week is followed, wherein the loading dose is 2 or 3 times greater than the about maintenance dose, and the antibody or fragment thereof is parenterally (e.g. For example, intravenous, subcutaneous or intramuscular).

In some embodiments, two loading doses of the antibody or fragment thereof are administered prior to the maintenance dose. In some embodiments, a single loading dose of the antibody or fragment thereof is administered per day, the two loading doses are administered, e.g., after about 1 or 2 weeks, and the maintenance dose is administered, e.g., 1 weekly during the treatment period. It is administered once or once every two weeks. In certain embodiments, the single loading dose is about 3 or 4 times greater than the maintenance dose, and the 2 loading dose is about 2 times greater than the maintenance dose. In some embodiments, the antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237, or 2A-F8) is about 3-15 mg, 15-30 mg or 30- 150 mg, or a single loading dose of about 4-20 mg, 20-40 mg or 40-200 mg, and about 2-10 mg, 10-20 mg or 20-100 mg after about 1 or 2 weeks. After administration in two loading doses, about 1-5 mg, 5-10 mg or 10-50 mg once weekly or once every two weeks during treatment (e.g., at least about 2, 3 or 6 months). A maintenance dose is followed, wherein if the single loading dose is about 3 or 4 times greater than the maintenance dose, the double loading dose is about 2 times greater than the maintenance dose, and the antibody or fragment thereof is parenterally (e.g., intravenous Intramuscularly, subcutaneously or intramuscularly).

In some embodiments, the one or more additional therapeutic agents are chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/ 208, 236/237 or 2A-F8).

The present disclosure provides for an MBG-related disease comprising administering to a subject in need of treatment a therapeutically effective amount of a chimeric, humanized or human anti-MBG antibody or fragment thereof described herein, optionally with an additional therapeutic agent. Provides a method of treatment. The present disclosure comprises a chimeric, humanized or human anti-MBG antibody or fragment thereof described herein, or a chimeric, humanized or human anti-MBG antibody or fragment thereof described herein for use as a medicament, optionally in combination with an additional therapeutic agent. It further provides a composition comprising. In addition, the present disclosure provides the use of a chimeric, humanized or human anti-MBG antibody or fragment thereof described herein, optionally in combination with an additional therapeutic agent, in the manufacture of a medicament. In some embodiments, the medicament is used for treatment of an MBG-related disease.

VI. Combination therapy with additional therapeutic agents

One or more additional therapeutic agents are optionally chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8 ) Can be used in combination. Any additional therapeutic agent(s) may be administered to the subject simultaneously with the antibody or fragment thereof (eg, in the same composition as the antibody or fragment thereof or in a separate composition) or sequentially (before and after).

In some embodiments, the optional additional therapeutic agent(s) is selected from MBG inhibitors, magnesium sulfate, blood pressure lowering agents, antidyslipidemic agents, antiplatelet agents, anticoagulants, anti-inflammatory agents, antioxidants, antifibrotic agents, and combinations thereof. do.

Examples of MBG inhibitors include, without limitation, resibufogenin (RBG), spironolactone, canrenone, and analogs, derivatives and salts thereof. Spironolactone is a competition antagonist of the ^{Na +} /K ^{+ -ATPase CTS binding site.} Canlenone is the main metabolite of spironolactone.

Magnesium sulfate may have an antihypertensive effect and may increase the activity of anti-MBG antibodies and fragments thereof. In addition, magnesium lowers high blood pressure in a drug-dependent manner.

In some embodiments, the one or more antihypertensive agents are chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 Or 2A-F8). Antihypertensive agents include, but are not limited to: MBG inhibitors, antagonists of the renin-angiotensin-aldosterone system (RAAS), diuretics, calcium channel blockers, α ₂ -adrenergic receptor agonists, α ₁ -adrenergic receptor antagonists (alpha blockers ), β-adrenergic receptor (β ₁ or/and β ₂ ) antagonists (beta blockers), mixed alpha/beta blockers, vasodilators, endothelin receptor antagonists, protein kinase C inhibitors (PKC exhibits vasoconstrictor effects on VSMC) , Inhibitors of the accumulation or effect of advanced glycosylation end products (AGE, inhibiting vasodilation by interfering with nitric oxide), minerals, and analogs, derivatives and salts thereof.

In a further embodiment, a chimeric, humanized or human anti-MBG antibody or fragment thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8) is used to treat a cardiovascular or cerebrovascular disorder. Hazardous anti-dyslipidemic agents, anti-platelet agents or anticoagulants, or any combination or both thereof. In certain embodiments, the anti-dyslipidemic agent comprises or is a statin (e.g., atorvastatin or a salt thereof) or/and fibrate (e.g., fenofibrate), and is an antiplatelet agent. Includes or is a COX-1 inhibitor (e.g., aspirin) or/and a P2Y ₁₂ inhibitor (e.g. clopidogrel), and the anticoagulant is a direct factor Xa inhibitor (e.g., apixaban or riva Rivaroxaban) or/and direct thrombin inhibitors (eg dabigatran). In addition, anti-dyslipidemic agents (e.g., statins such as atorvastatin or salts thereof) may be used to treat obesity-related diseases (e.g., metabolic syndrome or type 2 diabetes). For example, it can be used in combination with H2L2, H3L2, 201/202, 206/208, 236/237 or 2A-F8). In addition, statins have anti-inflammatory (eg, inhibit pro-inflammatory IL-6 and IL-8 secretion) and antioxidant (eg, reduce oxidative stress-induced cell damage) properties.

Anti-dyslipidemic agents include, but are not limited to: HMG-CoA reductase inhibitors, acetyl-CoA carboxylase (ACC) inhibitors, peroxisome proliferative activating receptor-alpha (PPAR-α) agonists, PPAR- Sterol O-acyl, including δ agonist, PPAR-γ agonist, liver X receptor (LXR) agonist, retinoid X receptor (RXR) agonist, acyl-CoA cholesterol acyltransferase (ACAT, ACAT1 [SOAT1] and ACAT2 [SOAT2]) Also known as transferase [SOAT]) inhibitor, stearoyl-CoA desaturase-1 (also known as SCD-1, stearoyl-CoA delta-9 desaturase) activity or expression inhibitor, cholesteryl ester transfer protein (CETP) inhibitor, microsomal triglyceride transfer protein (MTTP) activity or expression inhibitor, glucagon-like peptide-1 (GLP-1) receptor agonist, dipeptidyl peptidase 4 (DPP-4) inhibitor, pro-protein converting enzyme subtilis Sin/kecin type 9 (PCSK9) activity or expression inhibitors, apolipoprotein mimetic peptides, omega-3 fatty acids, and analogs, derivatives and salts thereof.

Antiplatelet agents include, without limitation: cyclooxygenase (e.g., COX-1) inhibitors, thromboxane (e.g., A ₂ ) synthase inhibitors, thromboxane (e.g., A ₂ ) receptors. Antagonists, adenosine diphosphate (ADP) receptor/P2Y ₁₂ inhibitors, adenosine reuptake inhibitors, glycoprotein IIb/IIIa inhibitors, phosphodiesterase (e.g., PDE3 or/and PDE5) inhibitors, protease activating receptor 1 (PAR1 ) Antagonists, prostacyclines and analogs thereof, and analogs, derivatives and salts thereof.

Anticoagulants include, without limitation, vitamin K antagonists, indirect factor Xa inhibitors, direct factor Xa inhibitors, direct thrombin (factor IIa) inhibitors (DTI) and analogs, derivatives and salts thereof.

Inflammation contributes to the pathophysiology of various disorders. Inflammatory disorders include, but are not limited to, autoimmune diseases (e.g., type 1 diabetes, multiple sclerosis and systemic sclerosis), high blood pressure, preeclampsia, edema, cardiovascular disease (e.g., atherosclerosis, myocardial ischemia/infarction, ischemia-reperfusion injury. [IRI] and peripheral arterial disease), brain disease (eg stroke and IRI), kidney disease (eg glomerulonephritis), liver disease (eg liver cirrhosis, nonalcoholic and alcoholic steatohepatitis, chronic Liver disease), eye disease (eg age-related macular degeneration [AMD]), metabolic syndrome, diabetes (type 1 and 2), sleep apnea (eg obstructive sleep apnea). In addition, inflammation is a major stimulant of fibrosis.

In some embodiments, the one or more anti-inflammatory agents are chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A It is used in combination with -F8). In certain embodiments, the one or more anti-inflammatory agents comprise or are inhibitors of pro-inflammatory cytokines or receptors therefor or production thereof (eg, TNF-α or/and IL-6 or IL-6R).

Anti-inflammatory agents include, but are not limited to: nonsteroidal anti-inflammatory agents (NSAIDs), immune modulators, immunosuppressants, anti-inflammatory cytokines and compounds that increase their production, inhibitors of pro-inflammatory cytokines or receptors therefor, pro -Inhibitors of the production of inflammatory cytokines or receptors therefor, pro-inflammatory transcription factors or inhibitors of their activation or expression, inhibitors of prostaglandins (eg prostaglandin E ₂ [PGE ₂ ]) or receptors therefor ( For example, EP ₃ ) or its production, leukotriene or its receptor or inhibitor of its production, phospholipase A2 inhibitor (e.g., secretion and cytoplasmic PLA2), C-reactive protein (CRP) activity or level inhibitor, Mast cell stabilizers, phosphodiesterase inhibitors, specialized pro-resolving mediators (SPM), other anti-inflammatory agents and their analogs, derivatives, fragments and salts.

Nonsteroidal anti-inflammatory agents (NSAIDs) include, without limitation: acetic acid derivatives, anthranilic acid derivatives (phenamate), enolic acid derivatives (oxicam), propionic acid derivatives, salicyl Rates, COX-2-selective inhibitors, other types of NSAIDs, such as monoterpenoids (eg eucalyptol and phenols [eg carbacrole]), anilinopyridinecarboxylic acids (E.g., clonixin), sulfonanilide (e.g., nimesulide), and lipooxygenase (e.g., 5-LOX) and cyclo Oxygenase (e.g. COX-2) (e.g., chebulagic acid, licofelone, 2-(3,4,5-trimethoxyphenyl)-4-( N-methylindol-3-yl)thiophene, and di-tert-butylphenol based compounds [eg, DTPBHZ, DTPINH, DTPNHZ and DTPSAL]); And analogs, derivatives and salts thereof.

The glucocorticoid class of corticosteroids has anti-inflammatory and immunosuppressive properties. Glucocorticoids include, without limitation, the hydrocortisone type, halogenated steroids, carbonates, and analogs, derivatives and salts thereof.

Also, for example, reactive oxygen species (ROS, such as superoxide radical, singlet oxygen, hydroxyl radical, peroxyl radical, hydrogen peroxide and organic hydroperoxide [eg, lipid hydroperoxide]), reactive nitrogen Oxidative stress, including species (RNS, such as nitric oxide radicals, nitrogen peroxide, nitrogen dioxide and dinitrogen trioxide) and radicals (eg, carbonate radicals), plays an important role in the pathophysiology of various diseases. For example, oxidative disorders characterized by oxidative stress or/and oxidative damage/injury include, without limitation, preeclampsia, cardiovascular disease (e.g., atherosclerosis, heart failure, myocardial ischemia/infarction and IRI), brain disorders (e.g. For example, stroke and IRI), neurodegenerative diseases (e.g., amyotrophic lateral sclerosis, multiple sclerosis and Alzheimer's, Huntington and Parkinson's disease), kidney disease (e.g. diabetic nephropathy), liver disease (e.g. , Cirrhosis), eye diseases (eg AMD), metabolic syndrome, diabetes (type 1 and 2), and sleep apnea (eg obstructive sleep apnea). In addition, oxidizing agents (eg, ROS and RNS) and oxidizing molecules (eg, oxidizing liquids) can be very inflammatory.

In some embodiments, the one or more antioxidants are chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or 2A- It is used in conjunction with F8). In certain embodiments, the one or more antioxidants are vitamins or analogs thereof (e.g., vitamin E or analogs thereof, such as α-tocopherol or trolox) or/and ROS, RNS or radical scavengers (e.g., Melatonin or/and glutathione).

Antioxidants include, without limitation; Vitamins and their analogs, carotenoids, sulfur-containing antioxidants, scavengers of ROS, RNS and radicals, enzyme inhibitors that produce ROS, substances that mimic or increase the activity or production of antioxidant enzymes, upregulate the expression of antioxidant enzymes. Transcription factor activators, mitochondrial target antioxidants, other types of antioxidants, and analogs, derivatives and salts thereof.

In a further embodiment, the one or more antifibrotic agents are chimeric, humanized or human anti-MBG antibodies or fragments thereof (e.g., H2L2, H3L2, 201/202, 206/208, 236/237 or It is used in combination with 2A-F8). In some embodiments, the one or more anti-fibrotic agents are anti-inflammatory agents or/and antioxidants (e.g., vitamin E or an analog thereof (e.g., α-tocopherol or trolox), sulfur containing antioxidants or ROS, RNS. Or radical scavenger [eg, melatonin], or any combination thereof). In certain embodiments, the at least one antifibrotic agent is pirfenidone (which also reduces fibroblast proliferation among the various antifibrotic and anti-inflammatory properties described herein) or/and nintedanib (fibroblast proliferation, migration and transformation. Blocking signaling of FGFR, PDGFR and VEGFR related to) or these.

In another embodiment, the at least one antifibrotic agent comprises or is one or more agents having anti-hyperglycemic or/and insulin-sensitizing activity for the treatment of a fibrotic disease in which hyperglycemia, diabetes or insulin resistance contributes to the development of fibrosis. . Examples of such disorders include, without limitation, diabetic kidney disease characterized by renal fibrosis, and nonalcoholic steatohepatitis (NASH) and cirrhosis, both characterized by liver fibrosis. The use of antihyperglycemic agents or/and insulin sensitizing agent(s) can reduce or prevent kidney inflammation and fibrosis or liver inflammation and fibrosis, for example. In certain embodiments, the at least one antifibrotic agent comprises or is a PPAR-γ agonist (eg, thiazolidinedione [above] such as pioglitazone or rosiglitazone). PPARγ-activated thiazolidinedione has both antihyperglycemic and insulin sensitization.

Antifibrotic agents include, but are not limited to: MBG inhibitors, collagen accumulation inhibitors, pro-fibrotic growth factors (eg, TGF-β [including TGF-β1], CTGF and platelet derived growth factors [PDGF-B, PDGF-). C and PDGF-D]) or their production, activation or signal transduction inhibitors, receptor tyrosine kinase (TK) inhibitors, anti-EGFR antibodies, anti-inflammatory agents including those listed above, antioxidants including those listed above, those listed above Antagonists of the renin-angiotensin-aldosterone system (RAAS), including advanced glycosylation end products (especially AGEs that increase arterial stiffness and stimulate intermediate matrix expansion), RGD mimetics and analogs (fibroblasts and immune cells on ECM glycoproteins) Inhibitors of the accumulation or effect of other types of antifibrotic agents, including, but not limited to, (e.g., NS-11, SF-6,5 and GRGDS), galectin-3 inhibitors (e.g. For example, GM-CT-01 and GR-MD-02), tricostatin A (inhibits TGFβ1-induced EMT), and PPAR-γ agonists (eg, thiazolidinidine [above]); And analogs, derivatives, fragments and salts thereof.

Any additional therapeutic agent(s) can be administered independently in any suitable mode. Potential modes of administration include, but are not limited to, oral, parenteral (including intradermal, subcutaneous, intramuscular, intravascular, intravenous, intraarterial, intraperitoneal, intramedullary, intrathecal and topical), intranasal and topical (skin/epidermis, Transdermal, mucosa, transmucosa, nasal cavity [eg, by nasal spray or drop], intraocular [eg, by eye drops], lung [eg, by oral or nasal inhalation], buccal, sublingual , Including rectal [eg, by suppository] and vaginal [eg, by suppository]). In some embodiments, any additional therapeutic agent(s) are administered independently orally or parenterally (eg, intravenously, subcutaneously or intramuscularly).

Any additional therapeutic agent(s) can be independently selected daily (once a day, twice or more), every two or three days, twice a week, once a week, every two weeks, every three weeks, every month, without limitation. It can be administered every month or every three months, or at any suitable frequency, including in an irregular manner or as needed. The frequency of administration may vary, for example, depending on the mode of administration selected. The duration of treatment with any additional therapeutic agent(s) can be determined by the treating physician and independently, e.g., at least about 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, 3 weeks, 4 It can be weeks (1 month), 6 weeks, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or more.

VII. Diagnostic use of anti-MBG antibodies and fragments thereof

The chimeric, humanized and human anti-MBG antibodies and fragments thereof disclosed herein are also useful in the diagnosis and prognostic evaluation of MBG-related diseases, including those associated with increased MBG levels (eg, hypertensive diseases). In addition, these antibodies and fragments thereof can be used to facilitate treatment decisions.

Anti-MBG antibodies or fragments thereof can be applied to detect the presence of MBG in an original or treated sample obtained from a subject. Biological samples can include, for example, blood, plasma, serum, urine, cerebrospinal fluid (CSF), cells or tissues. Samples can be analyzed directly, extracted prior to analysis, or expanded in volume by adding a suitable solvent.

In some embodiments, the biological sample is contacted with an anti-MBG antibody or fragment thereof, and the sample is screened to detect binding of the antibody or fragment thereof to MBG. Detection of this binding indicates the presence of MBG in the sample. In certain embodiments, the anti-MBG antibody or fragment thereof is labeled with a detectable agent (eg, a fluorescent dye), so that binding of the antibody or fragment thereof to MBG elicits a signal. In another embodiment, in the biological sample, the MBG is immobilized on the surface prior to introduction of the labeled anti-MBG antibody or fragment thereof (direct analysis), and the amount of signal corresponding to the amount of the labeled antibody or fragment thereof bound to the MBG is determined by the sample Is related to the amount of MBG. In another embodiment, the MBG in the biological sample is captured by an unlabeled first antibody immobilized on the surface and then bound to the captured MBG and to a labeled second antibody that generates a signal proportional to the amount of MBG captured. (Sandwich analysis), wherein the unlabeled first antibody and the labeled second antibody bind to different epitopes of MBG, and the unlabeled first antibody or/and labeled second antibody are independently used herein. It may be the disclosed anti-MBG antibody or fragment thereof.

In a further aspect, MBG in the biological sample is detected in a competition assay. In some embodiments, a sample (optionally suspended in a buffer) is mixed with a labeled anti-MBG antibody or fragment thereof. The resulting mixture is then contacted with the MBG-coated substrate. To facilitate MBG coating of a substrate (e.g., well of a microtiter plate), MBG is a carrier protein (e.g., bovine serum albumin [BSA], keyhole limpet hemocyn) that adsorbs onto the substrate, for example. May be conjugated to [keyhole limpet hemocyanin, KLH] or ovalbumin). The higher the amount of MBG in the sample, the more antibody/MBG complexes are formed, and fewer unbound (free) antibodies are available to bind to MBG on the substrate (“competition”), resulting in less signal.

In the direct, sandwich and competition assays described above, the primary anti-MBG antibody or fragment thereof can be labeled with a detectable agent. Alternatively, in direct, sandwich and competition assays, the primary anti-MBG antibody or fragment thereof may be unlabeled, and after binding of the primary antibody of MBG, the labeled secondary antibody (e.g., Antibody that binds to the Fc region). When a secondary antibody is conjugated to an enzyme, the addition of a substrate of the enzyme results in an enzyme/gazal reaction that produces a signal (eg, a chromogenic, fluorescent or electrochemical signal). The absorbance, fluorescence or electrochemical signal (e.g., current) of the solid support (e.g., plate well or bead) is measured to determine the presence and amount of MBG in the sample. Non-limiting examples of mustard horseradish peroxidase (HRP) substrates include 3-amino-9-ethylcarbazole (AEC), 3,3'-diaminobenzidine (DAB) and 3,3',5,5 '-Tetramethylbenzidine (TMB), examples of alkaline phosphatase include 5-bromo-4-chloro-3-indolyl phosphate (BCIP), examples of β-glucuronidase are 5- Bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). This assay is called enzyme-linked immunosorbent assay (ELISA).

Detectable agents include, without limitation, chromophores (eg, dyes, dyes, pigments and chromosomes (eg, 3-amino-9-ethylcarbazole [AEC], 5-bromo-4-chloro- 3-indolyl phosphate [BCIP], 3,3'-diamino benzidine [DAB]) and 3,3',5,5'-tetramethylbenzidine [TMB])}, fluorophore/fluorescent dye ( fluorochrome) (e.g. fluorescent dyes such as fluorescein, fluorescein isothiocyanate and rhodamine), chemiluminescent compounds (e.g. luciferin and luminol) , Radioactive isotopes (e.g. ³ H, ¹⁴ C, ³² P, ³⁵ S and ¹²⁵ I), radioactive elements (e.g. technetium), electron density compounds, magnetic materials and particles (e.g. , Paramagnetic and superparamagnetic substances), magnetic resonance imaging (MRI) contrast agents (eg, those containing gadolinium), enzymes (eg mustard radish oxidase [HRP], alkaline phosphatase, luciferase, β-glucuronidase and β-galactosidase), hapten and toxins.

The anti-MBG antibodies and fragments thereof described herein can be applied to a variety of immunoassay assays. These assays include, without limitation, chromogenic, fluorescence, chemiluminescence, light scattering, radiolabeling, electrochemistry, enzymes, precipitation, aggregation, coagulation, western blots, grid blots, tissue blots, and dots. Includes dot blot, dip stick and biosensor analysis. See, eg, Principles and Practice of Immunoassays, C. Price and D. Newman (Eds.), Stockton Press (1997); and The Immunoassay Handbook, 2ndEd. , D. Wild (Ed.), Nature Publishing Group (2001). In addition, anti-MBG antibodies and fragments thereof can be used for imaging such as MRI.

Detection and measurement of the amount of MBG in a biological sample can facilitate diagnosis, prediction and treatment of MBG-related diseases. In some embodiments, the disease is associated with increased levels of MBG, and an increased MBG level in a sample from a subject compared to the MBG level in a corresponding sample from another subject(s) without the disease is the diagnosis of the disease in the subject. Represents. The reference MBG level for diagnosis of a disease is, for example, a corresponding sample obtained from a statistically or epidemiologically significant number of subjects without a disease for the MBG level in a corresponding sample obtained from a statistically or epidemiologically significant number of subjects with a disease. It can be determined based on the comparison of the MBG level at. Likewise, for measurement and diagnosis, the level of MBG in a sample obtained from a subject can be used to determine the current severity of the disease and predict possible course or outcome (e.g., progression or regression) of the disease. It can be compared to a measure of the MBG level that is related to the severity of.

In addition, detection and measurement of the amount of MBG in a biological sample can facilitate treatment decisions. In some embodiments, the amount of the anti-MBG antibody or fragment thereof administered to the subject or/and the frequency of administration of the antibody or fragment thereof to the subject is maintained or adjusted (increased or decreased), or the antibody or fragment thereof to the subject is Discontinued based on the presence, absence, amount or level of MBG in the subject's sample.

In some embodiments, the kit uses an anti-MBG antibody or fragment thereof, which can be selectively labeled with a detectable agent, and optionally an anti-MBG antibody or fragment thereof for diagnostic applications (e.g., in an immunoassay). Includes instructions for

Blood MBG levels are elevated in hypertension and hypertension related diseases. For example, plasma MBG levels are 5 times higher in preeclampsia (PE) patients than in normal blood pressure pregnant women, and plasma MBG levels are associated with PE severity (Lopatin 1999 and Agunanne 2011). In addition, plasma MBG levels are increased 7-fold in patients with essential hypertension or congestive heart failure (CHF) and more than 70-fold in patients with chronic renal failure (Puschett, AJKD 2010). The severity of CHF is associated with plasma MBG levels (Fridman 2002). In addition, plasma MBG levels are increased in patients with myocardial ischemia/infarction (Bagrov 1998), plasma MBG levels are increased in patients with obstructive sleep apnea (OSA), and are associated with the severity of OSA (Zvartau 2006).

Other cardiotonic steroids, including other bufadienolides such as telocinobufagin (TCB) and resibufogenin (RBG), can also be a diagnosis of the disease. For example, plasma TCB levels are elevated in patients with chronic renal failure (Komiyama 2005). RBG blocks the effect of MBG or has the opposite effect of MBG (Puschett 2012 and Uddin, HP 2012).

Angiogenic imbalance induced by MBG contributes to PE (Uddin, TR 2012). Angiogenic balance is determined by anti-angiogenic factors (eg, soluble fms-like tyrosine kinase 1 [sFlt1 or sVEGFR1] and soluble endoglin [sEng]) and pro-angiogenic factors ( pro-angiogenic factor) (eg, vascular endothelial growth factor [VEGF] and placental growth factor [PlGF]). In PE patients, sFlt1 and sEng levels are increased prior to clinical symptoms and are also associated with PE severity, but PlGF levels are significantly reduced (Verlohren 2012 and Hagmann 2012). For example, excessive placental-derived sFlt1 contributes to endothelial dysfunction, hypertension and proteinuria in PE (Maynard 2003), and elevated levels of placental-derived sEng, a soluble TGF-b coreceptor, induces vascular permeability, hypertension and IUGR in PE. Do (Venkatesha 2006). At concentrations between 0.1 nM and 1 nM, MBG increases the expression of sFlt1 and sEng, and decreases the expression of VEGF and PlGF in the cell line of extrachronal CTB (Ehrig 2014). The sFLt1:PlGF ratio is particularly useful in determining the abnormal placental development and angiogenic imbalance characteristic of PE (Hertig 2010 and von Dadelszen 2014), and blood pressure during pregnancy is positively correlated with the sFlt1:PlGF ratio.

VIII. Preparation of anti-MBG antibodies and fragments thereof

The present disclosure provides polynucleotides comprising nucleic acid sequences encoding chimeric, humanized and human anti-MBG antibodies and fragments thereof described herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding _{the V H} domain or/and the V _{L domain of an anti-MBG mAb or fragment thereof.} In a further aspect, the polynucleotide comprises a nucleic acid sequence encoding the heavy or/and light chain of an anti-MBG mAb or fragment thereof.

The present disclosure further provides vectors (also referred to as expression or cloning vectors) comprising nucleic acid sequences encoding chimeric, humanized and human anti-MBG antibodies and fragments thereof described herein. Suitable vectors include plasmids, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, lambda phages (e.g., lysogeny gene deletions). One), and viruses. In certain embodiments, the vector is a plasmid.

Various vector systems can be applied. One class of vectors is adenovirus, baculovirus, bovine papilloma virus, polyoma virus, SV40 virus, vaccinia virus, and retroviruse (e.g. For example, DNA elements derived from animal viruses such as MMTV, MOMLV and Rous sarcoma viruses) are used. Another class of vectors uses RNA elements derived from RNA viruses such as eastern equine encephalitis virus, flaviviruse and Semliki Forest virus.

_{The vector may contain a variety of other elements for optimal expression of the mRNA in addition to, for example, the V H} domain or/and V _L domain of the anti-MBG mAb or fragment thereof, or the nucleic acid sequence encoding the heavy or/and light chain. . For example, the vector may be an open reading frame with or without a transcriptional promoter, promoter + operator, enhancer, intron(s) or/and exon(s), termination signal, splice signal. signal), a secretory signal sequence or selectable marker (eg, a gene that confers resistance to antibiotics or cytotoxic agents), or any combination or all thereof.

In addition, the present disclosure provides host cells comprising or expressing vectors encoding chimeric, humanized and human anti-MBG antibodies and fragments thereof described herein. Suitable host cells include eukaryotic cells, mammalian cells (e.g., BHK, CHO, COS, HEK293, HeLa, MDCKII and Vero cells), insect cells (e.g. Sf9 cells), yeast cells and bacterial cells (e.g. For example, E. coli cells), but is not limited thereto. In certain embodiments, the host cell is a mammalian cell (eg, a CHO cell or a HEK293 cell).

_{In some embodiments, the host cell comprises or expresses a vector encoding the V H} domain or V _L domain of the anti-MBG mAb or fragment thereof, or a heavy or light chain. In a further aspect, the host cell comprises _{or expresses a single vector encoding the V H} domain and V _L domain, or heavy and light chains of the anti-MBG mAb or fragment thereof. In another embodiment, the same host cell or a separate host cell is _{a vector encoding the V H} domain or heavy chain of the anti-MBG mAb or fragment thereof, and a separate vector encoding the V _L domain or light chain of the anti-MBG mAb or fragment thereof. It contains or expresses a vector.

Vectors can be transfected or introduced into host cells by any method known in the art. Transfection agents and methods include, without limitation, calcium phosphate, cationic polymers (e.g., DEAE-dextran and polyethyleneimine), dendrimers, fugenes, cationic liposomes, electroporation, sonoporation. ), cell compression, gene gun, viral transfection and retroviral transduction.

Methods and conditions for culturing the transfected host cell and recovering the recombinantly produced anti-MBG antibody or fragment thereof are known in the art and depend, for example, on the specific expression vector or/and host cell to be applied. Can be built or optimized. _{In some embodiments, the V H} domain or/and V _L domain, or heavy or/and light chain of the anti-MBG mAb or fragment thereof are produced recombinantly. In certain embodiments, the heavy and light chains of an anti-MBG whole IgG1, IgG2 or IgG4, or heavy and light chains of an anti-MBG Fab fragment optionally fused with an extension moiety are produced recombinantly.

IX. Cited literature

The following non-licensed documents are cited herein:

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All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and disclose and/or disclose methods and/or materials in connection with which the publication is cited. It is incorporated herein by reference to describe.

The following examples are for illustrative purposes only. Other assays, studies, processes, protocols, procedures, methodologies, reagents and conditions may alternatively be appropriately used.

Example

Example 1. Generation of chimeric anti-MBG antibodies

Chimeric antibodies were generated from mouse antibodies that bind to MBG with high specificity and high affinity. _{The V H} domain and V _L domain of the mouse antibody (SEQ ID NOs: 1 and 2) were fused to the _{human IgG2 C H} 1, C _H 2 and C _H 3 domains or the human kappa C _{L domain, respectively.}

Example 2. Generation of humanized anti-MBG antibody

Mouse antibodies that bind to MBG were also humanized. The CDRs of the heavy chain (IgG1) and light chain (kappa) of 3E9 (SEQ ID NOs: 3-8) were grafted to the receptor human framework sequence.

Human framework sequences were selected by aligning the mouse framework sequences with a database of human framework sequences to find the closest human homologue (generally about 65-70% sequence identity) for each chain. Human VH4-31 and VH4-59 were most homologous to the mouse framework sequence for the heavy chain, and human VK2-29 and VK2-30 were the most homologous to the mouse framework sequence for the light chain. VH4-59 and VK2-30 are selected as receptor human framework sequences for the _{V H} and V _L domains, respectively, because they have a high utilization rate in the human antibody repertoire and show a high conservation rate at critical framework positions compared to other human framework sequences. Became.

Three 3D models of the Fv region of the target antibody were constructed using three combinations of _{the V H} domain of 1T4K, 1ORS and 1DQD and the V _{L domain of 1TET.} In this model, the template matched the CDR length and standard structure to that predicted in the 3E9 sequence. The amino acid positions of the framework regions that differ between mouse and human sequences and that may affect antigen binding were identified. Three humanized heavy chain variable sequences (H1, H2 and H3) and two humanized light chain variable sequences (L1 and L2) were designed. The humanized heavy and light chain variable sequences had about 87-94% similarity to the human germline. Humanized antibodies generated from all 6 combinations of 3 humanized heavy chains and 2 humanized light chains-H1L1, H2L1, H3L1, H1L2, H2L2 and H3L2-, each heavy chain were human IgG2 or IgG4 C _H 1, C _H 2 , And each light chain comprising a _{C H} 3 domain and comprising a human kappa C _{L domain.}

Example 3. Generation of Phage-derived, Human Anti-MBG Antibodies

A human phagemid library with 1.2 x 10 ¹⁰ independent clones was panned against MBG conjugated to bovine serum albumin (BSA). As panning proceeds to the third enrichment for MBG-binding clones, phages are first competitive in antigen using "non-specific" cardiac steroids (CTS, typically ouabain in 1-10 μM free molecule). After elution, a second competitive elution with MBG at a single concentration (1-5 μM). For the fourth concentration, the second elution was a series of stepwise concentrations of free MBG (0.1, 0.5, 1.0 and 5.0 μM), and the clones of the final 5 μM elution were recovered for screening. In addition, phage remaining in the antigen after final competitive elution is recovered using triethylamine at 100 μM and screened for binders, which leads to, for example, 236/237 antibodies with very high affinity and specificity for MBG. .

About 660 individual clones representing 6 different fan-rich fractions were tested for binding to MBG in an enzyme linked immunosorbent assay (ELISA) identifying dozens of MBG specific clones. About 110 putative MBG-binding antibodies (Fab fragments expressed in phage) were further characterized by BstN1 fingerprinting, DNA sequencing, or/and MBG competitive ELISA to identify 14 unique antibodies that bind and compete with MBG in ELISA. Created. The relative CTS specificity of the 14 human antibodies is shown in Example 8 below. Seven of the 14 unique anti-MBG Fab fragments (201/202, 203/204, 205/207, 206/208, 236/237, 238/239 and 240/241) subcloned heavy chain variable domains, Whole human IgG1 was transformed, and the complete light chain contained in each phage clone was transformed into a mammalian expression vector for expression in HEK293 cells. Some of the phage-derived, human anti-MBG Fab fragments and whole IgG1 antibodies have a kappa C _L domain, while others have a lambda C _L domain.

Example 4. Recombinant production of chimeric anti-MBG antibodies

To generate the chimeric 3E9 IgG mAb, _{the DNA sequences encoding the V H} domain and V _L domain of the mouse antibody were cloned using the RACE-PCR cDNA approach and in a separate expression plasmid similar to the expression plasmid used for the humanized mAb. Each was fused with a DNA sequence encoding a human IgG2 C _H 1, C _H 2 and C _H 3 domain or a human kappa C _{L domain (see Example 6).} Chimeric mAbs were generated recombinantly using a procedure similar to that described for humanized mAbs in Example 6.

Example 5. Recombinant production of humanized anti-MBG antibodies

Plasmid encoding humanized heavy chain H1, H2 or H3 fused to human IgG2 or IgG4 C _H 1, C _H 2 and C _H 3 domains (see Example 2) and humanized light chain L1 or L2 fused to _{human kappa C L domain} A plasmid encoding (see Example 2) was prepared. HEK293F cells (Invitrogen) are cultured in serum-free medium, and 6 different plasmids encoding H1, H2 or H3, and plasmids encoding L1 or L2 (H1L1, H2L1, H3L1, H1L2, H2L2 and H3L2). Combination). Four days after transfection, the cell culture supernatant was harvested. Antibodies were purified by protein A affinity chromatography.

CHO cells encode humanized anti-MBG mAb (e.g., H1L1, H2L1, H3L1, H1L2, H2L2, H3L2 heavy chain (e.g., H1, H2 or H3) and light chain (e.g., L1 or L2). Were co-transfected with separate plasmids, grown in serum-free medium and selected for 2 weeks with 10 νg/ml Puromycin and 500 νg/ml G418 Single cells from a stable pool were transferred to 96 well plates by FACS. Colonies were grown after 2-3 weeks Antibody levels in culture medium were screened by Elisa High antibody expressing clones were expanded into 24 well plates, 6 well plates, and 30 ml shake culture Antibody production Levels are characterized in batch culture, antibody production levels of the top clones were> 300 mg/L.

Example 6. Recombinant production of human anti-MBG antibodies

HEK293F cells co-transfected with separate plasmids encoding the heavy and light chains of human anti-MBG mAb (e.g., 201/202, 206/208, 236/237 or 2A-F8) for 4 days in 200 mL culture. It was grown in serum-free medium. The culture supernatant was harvested and filtered sterilized. Antibodies were purified by protein A affinity chromatography.

CHO cells were co-transfected with separate plasmids encoding the heavy and light chains of human anti-MBG mAb (e.g., 201/202, 206/208, 236/237 or 2A-F8) and grown in serum-free medium. And, for 2 weeks, 10 νg/ml of Puromycin and 500 νg/ml of G418 were selected. Single cells in a stable pool were sorted into 96 well plates by FACS. Colonies were grown for 2-3 weeks. Antibody levels in the culture medium were screened by Elisa. High antibody expressing clones were expanded into 24 well plates, 6 well plates and 30 ml shake culture. The level of antibody production is characterized in batch culture. The level of antibody production in the top clones was> 300 mg/L.

Example 7. Antigen binding of humanized anti-MBG

Antigen binding of humanized anti-MGB was analyzed in direct Elisa assay. 2 νg/ml of MBG-conjugated BSA was coated on the Elisa plate. After blocking with 1% BSA, a 2-fold dilution series of humanized anti-MBG was added to the plate. After washing, bound antibody was detected by adding anti-human Fc HRP conjugate, and quantified by adding TMB substrate. OD absorption at 450 nm was measured on a microplate reader. Of the six humanized antibodies, H3L2 showed the highest binding affinity similar to that of the parent 3E9 antibody (FIG. 1B) (FIG. 1A ).

Example 8. Binding activity (avidity) and relative CTS specificity of anti-MBG antibody

The binding activity of anti-MBG antibodies to MBG was estimated by ELISA. The wells of the microtiter plate were coated with 2 νg/ml of MBG-bovine serum albumin (BSA) or MBG-keyhole limpet hemocyanin (KLH). Then, the wells were blocked with 1% BSA. Various concentrations (typically from about 0.0001 nM to about 25 nM) of test IgG antibody were added to the wells. Test IgG bound to immobilized MBG was detected with anti-human Fc, mustard radish oxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch). After washing the wells, an appropriate HRP reagent (eg, TMB ultra, Thermo Fisher) was added to the wells. Optical density (OD) at 450 nm was measured with a microplate reader. The concentration of the test antibody at the middle point of the binding curve is an estimate of the binding activity to MBG.

[Table 1] Estimated binding activity of anti-MBG antibody (K _{A ) (antibody concentration or EC 50} at 50% maximal binding to antigen)

The specificity (relative affinity) of anti-MBG antibodies to MBG compared to various cardiac steroids (CTS) was determined by competitive ELISA. MBG immobilized in the presence of MBG, resibufogenin (RBG), cinobufagin (CINO), ouabain (OUB) or digoxin (digoxin, DIG), free of each anti-MBG antibody as a competitor Was tested for binding to. A constant amount of anti-MBG antibody (typically 1-5 μg/mL) and various concentrations of each CTS (10 ^-12 M to 10 ^-5 M) were pre-incubated prior to addition to the MBG-BSA coated wells. . After washing of the wells, anti-human Fc, HRP-conjugated secondary antibody (at 1:5000 dilution) was added to the wells and incubated at 37° C. for 1 hour. After washing of the wells, an appropriate HRP reagent (eg, TMB ultra, Thermo Fisher) was added to the wells. OD at 450 nm was measured with a microplate reader. At 10 μM, MBG was assumed to compete 100%, and at 1 pM, MBG was assumed to compete 0%. Data were plotted as titration of antibody-antigen binding to the free CTS concentration in a competition reaction to determine the free CTS concentration (μM) that gives 50% maximal binding of the antibody to the immobilized MBG. The relative specificity of the antibody for CTS was normalized to MBG = 100%.

[Table 2] The relative specificity of anti-MBG antibodies to free CTS competing with immobilized MBG (normalized to MBG = 100%), wherein the free which provides 50% maximal binding of the antibody to MBG-BSA The CTS concentration (μM) was determined.

* 10 μM of free CTS was insufficient to see the free CTS concentration giving 50% maximal binding of the antibody to MBG-BSA. When 236/237 was tested for competition with 100 μM free MBG or RBG, a complete binding curve was generated. 238/239 and 240/241 were not tested in 100 μM free CTS.

nc = not countable in competitive analysis/chart.

nt = not tested.

The three antibodies, 236/237, 2A-F8 and H3L2, did not react with the CTS competing molecule. Two antibodies, 201/202 and R4CE-1E, reacted with the CTS competitors RBG and CINO. Two antibodies, 203/204 and 206/208, reacted with the CTS competitor RBG. One antibody, 2A-5A, reacted with CTS competitors RBG, CINO and DIG. Two antibodies, 238/239 and 1B-6E, reacted with the CTS competitor OUB (but not tested against the other CTS competitor).

Example 9. Inhibition of MBG-induced PKC-δ nuclear translocation by anti-MBG Abs

MBG induces fibrosis including renal fibrosis, cardiac fibrosis and vascular fibrosis (Federova 2009, Elkareh 2007 and Nikitina 2011). MBG binding to NKA induces the translocation of PKC-δ from the cytosol to the nucleus, phosphorylating the transcription factor Fli-1, making Fli-1 proteolytic easy. Proteolysis of Fli-1 releases inhibition of the expression of procollagens I and III (Elkareh 2009). Increased collagen synthesis promotes fibrosis.

MBG was incubated with anti-MBG antibody prior to addition to cultured primary rat cardiac fibroblasts. The nuclei were separated after 30 minutes and analyzed for the amount of PKC-δ. Mouse anti-MBG mAb 3E9 served as a positive control, and non-MBG binding human IgG mAb served as a negative and isotype control.

Figure 2 is a Western blot showing the relative amounts of PKC-δ (78 kDa) in the nuclei of primary cardiac fibroblasts cultured with MBG alone or with MBG and antibodies. At 2 nM, MBG induced an increase in the amount of PKC-δ in the nucleus compared to “control” (treated with only vehicle, lane 1 in blot), and anti-MBG mAb 3E9, H3L1, 201/202, 206/208 And treatment with 236/237, and to a lesser extent with H2L1, H2L2, 238/239 and 240/241 (FIG. 2A ). Western blot of Figure 2b shows that the humanized 3E9 mAb H3L2 and all 6 phage derived human mAbs (two numbers with slash) reduced PKC-δ translocation from 10 nM MBG to the nucleus.

Example 10. Reversal of MBG inhibition of HUVEC cell proliferation by anti-MBG Abs

Endothelial dysfunction causes pathological features of preeclampsia, including increased vascular permeability/leakage (Puschett, BBA 2010). MBG promotes endothelial dysfunction in part by inhibiting endothelial cell proliferation (Uddin, Placenta 2008).

MBG (2nM) was incubated with antibodies prior to addition to cultured human umbilical vein endothelial cells (HUVEC) (20 μg/mL in FIG. 3A with human anti-MBG mAb, or with humanized anti-MBG mAbs. Various concentrations in 3b). After 48 hours, DNA synthesis was evaluated by incorporation of ^{3 H-thymidine.}

HUVEC cells in endothelial cell growth medium were plated in 96-well plates. The next day, the cells were pretreated with various antibodies (2.5-20 μg/mL), and then incubated with 2 nM MBG for 48 hours. ^[3 H] - thymidine was added to each well. [ ³ H]-thymidine labeled DNA was analyzed by ^{harvesting cells and analyzing for 3} H using a PerkinElmer Wallac Tailux liquid scintillation counter. Mouse anti-MBG mAb serves as a positive control, and non-MBG binding human IgG mAb serves as a negative and isotype control. Means were tested for significance using Student's t-test.

MBG inhibition of HUVEC cell proliferation was significantly reduced by 201/202, 236/237 and 238/239 among phage-derived human mAbs (FIG. 3A) and by H2L2 and H3L2 among humanized 3E9 mAbs (FIG. 3B ).

Example 11. Reversal of MBG-induced anti-proliferation and anti-angiogenic signals in CTB cells by anti-MBG antibody

MBG inhibits the proliferation, migration and invasion of cytotrophic blast (CTB) cells (Uddin, JBC and Placenta 2008). In addition, angiogenic imbalance induced by MBG promotes endothelial dysfunction in preeclampsia (Puschett, BBA 2010 and Uddin, TR 2012). In PE patients, sFlt1 and sEng levels increase prior to clinical symptoms and are also associated with PE severity, but PlGF levels are significantly reduced (Verlohren 2012 and Hagmann 2012). MBG induces anti-angiogenic signaling in CTB cells (Ehrig 2014).

CTB cells were serum depleted in the medium, washed twice, and the replicates were treated for 48 hours with DMSO (carrier) or medium containing 0.1, 1, 10 or 100 nM MBG (promoting cell proliferation). Some cells were pretreated with 30% molar excess of antibody 206/208, H1L2 or 3E9 before adding MBG. Culture media were collected for angiogenesis and analysis of anti-angiogenic factors. Cell viability was measured using CellTiter Assay (Promega; Uddin, JBC 2008 and Placenta 2008).

In a series of further experiments, human CTB cells were treated with DMSO (carrier) or 0.1, 1, 10 or 100 nM of MBG for 48 hours. Some cells were pretreated with 30% molar excess of antibodies 201/202, 206/208, 236/237, H1L2 or 3E9, while others were co-treated with antibodies and MBG. Mouse anti-MBG mAb was used as a positive control, and non-MBG-binding human IgG mAb was used as a negative and isotype control. Culture media were collected for analysis of angiogenesis and secretion of anti-angiogenic factors. Levels of soluble fms-like tyrosine kinase 1 (sFlt1 or sVEGFR1), soluble endoglin (sEng), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in the cell medium were measured in an ELISA kit (Boster Biotech, Boster Biotech, Pleasanton). , California). Cell lysates were analyzed for expression of proliferating cell nuclear antigen (PCNA) and phosphorylation of p38 MAPK by western blot. Cell titer assay (Promega) was used to measure cell viability. Statistical analysis was performed using analysis of variance and Duncan's post-test. A p value ≤ 0.05 was considered statistically significant.

Compared to vehicle (DMSO) treatment, treatment of CTB cells with ≥ 1 nM MBG markedly downregulated PCNA expression, increased phosphorylation of p38 MAPK, and significantly increased the secretion of anti-angiogenic factors sFlt1 and sEng. , Significantly reduced the secretion of angiogenic factors VEGF and PlGF (p <0.05, respectively). Pretreatment and co-treatment of CTB cells with each of the four test anti-MBG mAb and mouse antibodies significantly upregulated PCNA expression, reduced phosphorylation of p38 MAPK, significantly reduced the secretion of sFlt1 and sEng, and VEGF. And significantly increased the secretion of PlGF (p<0.05, respectively). All four test anti-MBG mAbs attenuated MBG induced anti-proliferative and anti-angiogenic signals in cultured CTB cells to a similar degree to murine anti-MBG mAb 3E9.

Example 12. DOCA/saline rat model of preeclampsia (PE)

In humans, the volume of extracellular fluid (ECF) increases by about 40-50% during pregnancy, and volume expansion promotes the secretion of MBG and ultimately the development of PE (Maynard 2001). In the volume expansion model of PE in pregnant rats (Vu 2005 and Puschett, BBA 2010), replacement of saline instead of normal drinking water with an aldosterone precursor desoxycorticosterone acetate (DOCA) injection resulted in volume expansion and increased urine MBG levels. And induced many phenotypic traits of human PE, such as hypertension, proteinuria, IUGR, decreased pups, and total pups weight loss (Vu 2005, Ianosi-Irimie 2005 and Horvat 2010).

Female Sprague-Dawley (SD) rats (200-250 g, Charles River Laboratories) were acclimated for 1 week and mated with male Sprague-Dawley (SD) rats (275-300 g). Pregnancy was confirmed by the presence of a vaginal plug or by a vaginal smear. Each rat was stored separately.

Five rat groups were studied (n = 10 per group): Group 1 (NP): normal pregnant rats; Group 2 (PDS): Pregnant rats initially receiving 12.5 mg of DOCA in depot form i.p, followed by weekly injection of 6.5 mg of DOCA i.p, and drinking water replaced with 0.9% saline; Group 3 (NPM): normal pregnant rats injected with MBG (7.65 μg per kg body weight per day) daily when pregnancy was confirmed on day 4 of the experiment; Group 4 (PDS-3E9): Rats administered DOCA and saline as in Group 2 and given murine anti-MBG antibody 3E9 (2.2 mg per kg body weight per day) on days 16, 17 and 18 of gestation; And Group 5 (PDS-H3L2): Rats administered DOCA and saline for Group 2 and given the humanized anti-MBG antibody H3L2 (2.2 mg/kg body weight per day) on days 16, 17 and 18 of gestation.

Systolic blood pressure (BP) was measured by tail cuff on days 17-19. After the BP stabilized, at least three readings were taken and the average of these values was calculated. The reported BP value is the average of the daily measurements. On the 18-20th day of gestation, urine was collected for 24 hours in the absence of food to suppress contamination of the urine by measuring the amount of protein in the urine by any fallen food particles. Each rat was stored separately in metabolic cages during this part of the study. Protein excretion was measured for 24 hours and normalized with creatinine. Rats were sacrificed after the last measurement on days 18-20 and blood samples were taken. Fetal offspring were counted and examined. Average number of pups and developmental or histological abnormalities of pups were assessed. Statistical analysis was performed using analysis of variance and Tukey's post-test. The p value ≤0.05 was considered statistically significant.

[Table 1]

DOCA and saline treatment increased blood pressure, proteinuria, overall litter size reduction and fetal malformations in pregnant rats. Administration of MBG alone showed similar effects. Anti-MBG and humanized anti-MBG antibodies prevented this increase in blood pressure, proteinuria and fetal malformations.

Example 13. Dahl salt sensitive rat model of preeclampsia

Dahl salt-sensitive (Dahl-S) rats spontaneously display many of the phenotypes characteristic of human PE during pregnancy, including hypertension, severe proteinuria exacerbation, renal changes such as glomerular hypertrophy, increased fetal mortality and decreased neonatal size (Gillis 2015) . In addition, pregnant Dahl-S rats have high levels of MBG.

Female Dahl-S rats (SS/jr, 200-250 g, Charles River) were acclimated for 1 week and crossed with male Dahl-S rats (275-300 g). Pregnancy was confirmed by the presence of a vaginal plug or by a vaginal smear. Each rat is stored separately.

Six rat groups were studied (n = 10 per group): Group 1: Non-pregnant female Dahl-S rats; Group 2: Pregnant Dahl-S rats; Group 3: Pregnant Dahl-S rats given an isotype and non-MBG binding human mAb (IgG) as a negative control twice weekly (i.p.) from day 10 to day 20 of gestation; Group 4: Pregnant Dahl-S rats administered with anti-MBG antibodies (eg H3L2, 236/237 or 2A-F8) twice weekly i.p. from day 10 to day 20 of gestation; Group 5: rats as same as group 4, but given 2 mg/kg of anti-MBG antibody twice weekly i.p. from day 10 to day 20 of gestation; And Group 6: Rats as in Group 4, but given 0.5 mg/kg of anti-MBG antibody twice weekly i.p. from day 10 to day 20 of gestation.

Systolic blood pressure was measured and calculated, the amount of protein in urine was measured for 24 hours on days 18-20 of gestation, and other measurements and evaluations were performed similarly to those described above for DOCA/saline rats. Statistical analysis was performed using analysis of variance and Tukey's post-test, where p value ≤ 0.05 indicates statistical significance.

Systolic blood pressure (BP) is measured on days 0 (baseline, T0), 8-10 days (T1) and 18-20 days (T2). After the BP has stabilized, at least three readings are taken daily and the average of these values is calculated. BP values reported for T0, T1 and T2 are averages of daily measurements.

On the 18th-20th day of pregnancy, urine was collected for 24 hours in the absence of food to suppress contamination of the urine by measuring the amount of protein in the urine by any fallen food particles. Each rat was stored separately in metabolic cages during this part of the study. Urine MBG, RBG and TCB levels were measured in a competition ELISA, and protein excretion was measured for 24 hours. Rats were sacrificed on days 18-20 and blood samples were taken. The pups were counted and examined. Average number of pups and developmental or histological abnormalities of pups were assessed.

Statistical analysis was performed using analysis of variance and Tukey's post-test, where p value ≤ 0.05 indicates statistical significance.

Example 14. STZ-CD1 mouse model of advanced diabetic kidney disease

CD1 mice were given streptozotocin (STZ) (200mg/kg) in a single i.p. By injection, type 1 diabetes (T1D) was induced as a result of the toxicity of STZ to insulin-producing pancreatic β-cells. STZ-CD1 T1D mice developed chronic kidney disease associated with decreased renal function and tubular interstitial fibrosis within 6 months after STZ injection (Sugimoto 2007). STZ-CD1 mice exhibited severe tubular interstitial fibrosis, moderate interstitial stromal dilatation, and moderate albuminuria (Kitada 2016).

T1D was induced by i.p. injection of vehicle or STZ (in 10 mM sodium citrate, pH 4.5, 200 mg/kg) into 8-week-old CD1 mice (7-week-old Charles River). Three days after STZ injection, T1D was confirmed by urine dipstick test. Each mouse was stored separately in metabolic cages. Mice were randomly divided into 6 groups (n = 12 per group) and treated at 1 month after STZ injection starting with i.p. twice weekly for 2 months as follows: Group 1: CD1 mice + vehicle only; Group 2: STZ-CD1 T1D mice + normal human IgG (10 mg/kg) as negative and isotype controls; Group 3: STZ-CD1 T1D mice + murine anti-MBG mAb 3E9 as positive control; Group 4: STZ-CD1 T1D mice + test anti-MBG antibody (eg, H3L2, 236/237 or 2A-F8) (1 mg/kg); Group 5: STZ-CD1 T1D mice + test anti-MBG antibody; And Group 6: STZ-CD1 T1D mice + test anti-MBG antibody. Mice were sacrificed 3 months after STZ injection.

With regard to the evaluation of clinical parameters, systolic blood pressure by tail plethysmography was monitored weekly. Urine samples were collected for 24 hours during the last week of treatment. Urine albumin was measured by ELISA (Exocell, Philadelphia, Pennsylvania). Serum or urine creatinine was determined by high pressure liquid chromatography (HPLC). Creatinine was quantified by comparison to a standard curve of pure creatinine (10-50 ng).

For histopathological analysis, perfused kidneys were removed and fixed overnight in formalin, and sections were stained with periodic acid-Schiff. Slides were analyzed to assess the extent of glomerular expansion and hardening. Each glomerulus in a single section is rated from 0 to 4, where 0 indicates no lesion and 1, 2, 3 and 4 are glomerular stroma comprising <25, 25-50, 50-75 or> 75% Indicative of sclerosis or sclerosis. Tubular interstitial fibrosis was reported by Zhang et al . (2012) was evaluated using picrosirius staining.

For immunohistochemistry, pre-fibrotic mediators (e.g., TGF-b and CTGF) and pro-inflammatory cytokines (e.g., TNF-a, IL-6 and MCP) of the kidney in cryopreserved sections stained with indirect immunofluorescence -1) was detected, and Kato et al . It was semi-quantified as described in (1999).

Statistical analysis was performed using analysis of variance and Tukey's post-test. A p value ≤ 0.05 is considered statistically significant.

Example 15. ZDF rat model of advanced diabetic kidney disease

Zucker diabetic fat (ZDF, Lepr ^fa /Crl) rat is a spontaneous model of DN. ZDF rats derived from the ZF strain show obesity with diabetes and are widely used in type 2 diabetes (T2D) studies. ZDF rats show gradual insulin resistance and glucose intolerance at 3-8 weeks of age, and become apparently diabetic at 8-10 weeks of age. ZDF rats did not show glomerulosclerosis, tubular cell damage or tubular interstitial fibrosis at 8 weeks, but the glomeruli of ZDF rats appeared slightly enlarged at 8 weeks compared to control lean rats. ZDF rats show an additional increase in glomerular lesions at 8 weeks of age, and moderate to severe interstitial stromal dilatation after 22 weeks. In addition, coronary cell damage, moderate to severe tubular interstitial fibrosis, inflammation and hypoxia are observed in the kidneys of ZDF rats at 22-39 weeks. ZDF rats exhibit moderate to severe interstitial stromal dilatation, moderate to severe tubular interstitial fibrosis, and moderate to severe albuminuria (Kitada 2016).

Male ZDF rats (10 weeks old, Charles River) and male lean (ZL) control rats (10 weeks old, Charles River) were stored separately in metabolic cages. Starting at week 15, rats were monitored for elevated blood glucose levels under fasting conditions. Diabetes is defined as a blood glucose level of> 250 mg/dL. Proteinuria was measured by ELISA (Exocell). Only hyperglycemic rats with severe albuminuria were placed in the treatment group and treated. Severe kidney pathology in ZDF rats is expected to become evident at 22 weeks of age (Chander 2004).

Rats were randomly divided into 6 groups (n = 5 per group) and treated with i.p. twice weekly after the onset of diabetes as follows: Group 1: ZL rats + carrier only; Group 2: ZDF diabetic rat + normal human IgG as negative and isotype control; Group 3: ZDF diabetic mice + murine anti-MBG mAb 3E9 (10 mg/kg) as positive control; Group 4: ZDF diabetic mice + test anti-MBG antibody (eg, H3L2, 236/237 or 2A-F8) (1 mg/kg); Group 5: ZDF diabetic mice + test anti-MBG antibody; And Group 6: ZDF diabetic mice + test anti-MBG antibody. Rats were sacrificed at the time of signs of death or at 39 weeks of age.

Evaluation of clinical parameters, histopathological analysis, immunohistochemistry and statistical analysis were performed similarly as described above for the STZ-CD1 mouse model of DN.

Example 16. Anti-MBG antibody is Na ⁺ K ⁺ -Prevents ATPase from inhibiting MBG.

Na ⁺ K ⁺ -ATPase (NKA) was purified from rat heart. NKA was collected as a sarcolemmal/particulate (SLP) fraction and frozen.

Na ⁺ K ⁺ -ATPase activity was measured as follows: SLP solution was subjected to reaction buffer (200mM Tris-HCl, pH 7.5, 200mM NaCL, 30mM MgCl ₂ , 60mM KCl, 10mM EGTA, 100 µg/mL PMSF, 2 µg/ mL pepstatin A, and 2 μg/mL aprotinin). Reactions were performed without MBG and with or without MBG antibody (1-100 μg/mL). Uavine was used as a positive control for NKA inhibition. The tube was heated in advance and ATP was added. The reaction was stored and placed on ice.

Phosphate was analyzed using Malachite Green Phosphate Assay. Reagent A was added after adding a phosphate standard or sample to each well of a 96 well plate. Reagent B was added, mixed and cultured. Thereafter, the OD was read at 630 nm on a microplate spectrophotometer. Uavine or MBG could significantly inhibit NKA activity. Anti-MBG mouse antibody 3E9 and humanized antibody H3L2 were found to reduce the inhibitory effect of MBG on NKA.

Example 17. Measurement of MBG in biological samples

Abi-Ghanem et al. J Immunoassay Immunochem. MBG levels were measured in urine by a modified competitive ELISA from 32:31-46 (2011). Wells were coated with anti-MBG antibody. Thereafter, samples containing MBG (known or unknown) and MBG-BSA were added to the wells. After HRP-anti-BSA antibody was added to each well, it was reacted with TMB (K&P Labs). The reaction was stopped and the wells were read at 450 nm. A standard curve was made using samples with known amounts of MBG (Figure 5). Urine samples were analyzed and compared to a standard curve. The concentration of MBG in urine samples was calculated from the linear range of the standard curve. The amount of MBG found in urine samples was similar to the values reported in the literature. For example, human urine sample #3 was analyzed twice to obtain OD values of 0.140 and 0.149. From the standard curve, these values corresponded to 224.6 +/- 23.7 pg/mL MBG. This example used a urine sample to measure MBG, but any biological fluid/sample can be used in the assay.

While certain embodiments are illustrated and described, it is understood that various modifications may be made herein and are contemplated herein. Further, it is understood that the present disclosure is not limited by the specific embodiments provided herein. Descriptions and examples of aspects of the present disclosure and examples herein are not intended to be construed in a limiting sense. It is further understood that all aspects of the present disclosure are not limited to the specific depictions, configurations, or relative proportions described herein, which may depend on various conditions and variables. Various modifications and variations in form and detail of aspects and embodiments of the present disclosure will be apparent to those skilled in the art. Accordingly, this disclosure is also contemplated to cover any and all such modifications, variations and equivalents.

SEQUENCE LISTING <110> Panorama Research Institute <120> Anti-Marinobufagenin Antibodies and Uses Thereof <130> PRI.0002 <160> 103 <170> PatentIn version 3.5 <210> 1 <211> 117 <212> PRT <213> Mus musculus <400> 1 Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Tyr Gly Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 2 <211> 112 <212> PRT <213> Mus musculus <400> 2 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 3 <211> 9 <212> PRT <213> Mus musculus <400> 3 Asp Ser Ile Thr Ser Gly Tyr Trp Asn 1 5 <210> 4 <211> 16 <212> PRT <213> Mus musculus <400> 4 Tyr Ile Ser Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 5 <211> 9 <212> PRT <213> Mus musculus <400> 5 Ala Arg Tyr Gly Tyr Gly Phe Asp Tyr 1 5 <210> 6 <211> 16 <212> PRT <213> Mus musculus <400> 6 Arg Ser Ser Gln Ser Ile Val His Ser Asn Arg Asn Thr Tyr Leu Glu 1 5 10 15 <210> 7 <211> 7 <212> PRT <213> Mus musculus <400> 7 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 8 <211> 9 <212> PRT <213> Mus musculus <400> 8 Phe Gln Gly Ser His Val Pro Leu Thr 1 5 <210> 9 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Humanized VH sequence <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Tyr Gly Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Humanized VH sequence <400> 10 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Tyr Gly Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 11 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Humanized VH sequence <400> 11 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ala Arg Tyr Gly Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 12 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized VL sequence <400> 12 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 13 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Humanized VL sequence <400> 13 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 14 <211> 122 <212> PRT <213> Homo sapiens <400> 14 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Ala Arg Tyr Asn Trp Asn Tyr Val Leu Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 111 <212> PRT <213> Homo sapiens <400> 15 Gln Ala Val Leu Thr Gln Pro Ser Ser Val Ser Ala Ala Pro Arg Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ala Ser Asn Ile Gly Arg Asn 20 25 30 Gly Val Ser Trp Tyr His Gln Val Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45 Leu Ser His Asp Gly Leu Val Thr Ser Gly Val Pro Asp Arg Leu Ser 50 55 60 Val Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu His 65 70 75 80 Ser Asp Asp Glu Gly Asp Tyr Tyr Cys Ala Val Trp Asp Asp Ser Leu 85 90 95 Asn Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser 100 105 110 <210> 16 <211> 10 <212> PRT <213> Homo sapiens <400> 16 Gly Gly Ser Ile Ser Ser Ser Ser Tyr Tyr 1 5 10 <210> 17 <211> 7 <212> PRT <213> Homo sapiens <400> 17 Ile Tyr Tyr Ser Gly Ser Thr 1 5 <210> 18 <211> 14 <212> PRT <213> Homo sapiens <400> 18 Ala Arg Ala Arg Tyr Asn Trp Asn Tyr Val Leu Phe Asp Tyr 1 5 10 <210> 19 <211> 8 <212> PRT <213> Homo sapiens <400> 19 Ala Ser Asn Ile Gly Arg Asn Gly 1 5 <210> 20 <211> 3 <212> PRT <213> Homo sapiens <400> 20 His Asp Gly One <210> 21 <211> 11 <212> PRT <213> Homo sapiens <400> 21 Ala Val Trp Asp Asp Ser Leu Asn Ala Val Val 1 5 10 <210> 22 <211> 120 <212> PRT <213> Homo sapiens <400> 22 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Ala Ser Ile Ala Ala Arg Thr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 110 <212> PRT <213> Homo sapiens <400> 23 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 24 <211> 10 <212> PRT <213> Homo sapiens <400> 24 Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr 1 5 10 <210> 25 <211> 7 <212> PRT <213> Homo sapiens <400> 25 Ile Tyr Tyr Ser Gly Asn Thr 1 5 <210> 26 <211> 12 <212> PRT <213> Homo sapiens <400> 26 Ala Arg Ala Ser Ile Ala Ala Arg Thr Phe Asp Tyr 1 5 10 <210> 27 <211> 8 <212> PRT <213> Homo sapiens <400> 27 Ser Ser Asn Ile Gly Asn Asn Tyr 1 5 <210> 28 <211> 3 <212> PRT <213> Homo sapiens <400> 28 Asp Asn Asn One <210> 29 <211> 11 <212> PRT <213> Homo sapiens <400> 29 Gly Thr Trp Asp Ser Ser Leu Ser Ala Tyr Val 1 5 10 <210> 30 <211> 118 <212> PRT <213> Homo sapiens <400> 30 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Gln Leu Lys Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Arg Pro Pro Ala Ala Thr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 31 <211> 110 <212> PRT <213> Homo sapiens <400> 31 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Ser Ile Ser Cys Ser Gly Gly Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Glu Asn Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Arg Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 32 <211> 8 <212> PRT <213> Homo sapiens <400> 32 Gly Gly Ser Ile Ser Ser Tyr Tyr 1 5 <210> 33 <211> 12 <212> PRT <213> Homo sapiens <400> 33 Ala Arg Val Arg Pro Pro Ala Ala Thr Phe Asp Tyr 1 5 10 <210> 34 <211> 3 <212> PRT <213> Homo sapiens <400> 34 Glu Asn Asn One <210> 35 <211> 11 <212> PRT <213> Homo sapiens <400> 35 Gly Thr Trp Asp Ser Ser Leu Arg Ala Val Val 1 5 10 <210> 36 <211> 119 <212> PRT <213> Homo sapiens <400> 36 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Val Asn Leu Arg Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 37 <211> 111 <212> PRT <213> Homo sapiens <400> 37 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Glu Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu 65 70 75 80 Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser 85 90 95 Leu Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 38 <211> 11 <212> PRT <213> Homo sapiens <400> 38 Ala Arg Val Asn Leu Arg Gly Ala Phe Asp Ile 1 5 10 <210> 39 <211> 9 <212> PRT <213> Homo sapiens <400> 39 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 40 <211> 11 <212> PRT <213> Homo sapiens <400> 40 Gly Thr Trp Asp Ser Ser Leu Ser Ala Val Val 1 5 10 <210> 41 <211> 125 <212> PRT <213> Homo sapiens <400> 41 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Val Val Val Ala Arg Thr Gly Thr Thr Ile Ser Trp Phe 100 105 110 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 42 <211> 115 <212> PRT <213> Homo sapiens <400> 42 Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg Ser Gly Ile Asn Val Asp Thr 20 25 30 Tyr Arg Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Gln Tyr 35 40 45 Leu Leu Arg Tyr Lys Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile 65 70 75 80 Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90 95 Val Ile Trp His Ser Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu 100 105 110 Thr Val Leu 115 <210> 43 <211> 17 <212> PRT <213> Homo sapiens <400> 43 Ala Arg Val Val Val Ala Arg Thr Gly Thr Thr Ile Ser Trp Phe Asp 1 5 10 15 Pro <210> 44 <211> 9 <212> PRT <213> Homo sapiens <400> 44 Ser Gly Ile Asn Val Asp Thr Tyr Arg 1 5 <210> 45 <211> 7 <212> PRT <213> Homo sapiens <400> 45 Tyr Lys Ser Asp Ser Asp Lys 1 5 <210> 46 <211> 9 <212> PRT <213> Homo sapiens <400> 46 Val Ile Trp His Ser Ser Ala Trp Val 1 5 <210> 47 <211> 121 <212> PRT <213> Homo sapiens <400> 47 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Leu Arg Tyr Ser Ser Gly Trp Tyr Ala Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 48 <211> 110 <212> PRT <213> Homo sapiens <400> 48 Leu Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Ile Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asn Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Ala Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Val Ala Trp Asp Gly Ser Leu 85 90 95 Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 49 <211> 13 <212> PRT <213> Homo sapiens <400> 49 Ala Arg Leu Arg Tyr Ser Ser Gly Trp Tyr Ala Asp Pro 1 5 10 <210> 50 <211> 8 <212> PRT <213> Homo sapiens <400> 50 Ser Ser Asn Ile Gly Ser Asn Ile 1 5 <210> 51 <211> 3 <212> PRT <213> Homo sapiens <400> 51 Asn Asn Asn One <210> 52 <211> 11 <212> PRT <213> Homo sapiens <400> 52 Val Ala Trp Asp Gly Ser Leu Ser Gly Val Val 1 5 10 <210> 53 <211> 121 <212> PRT <213> Homo sapiens <400> 53 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Phe His Thr Trp Ala Pro Arg Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 54 <211> 109 <212> PRT <213> Homo sapiens <400> 54 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Glu Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 55 <211> 13 <212> PRT <213> Homo sapiens <400> 55 Ala Arg Phe His Thr Trp Ala Pro Arg Ala Phe Asp Ile 1 5 10 <210> 56 <211> 10 <212> PRT <213> Homo sapiens <400> 56 Gly Thr Trp Asp Ser Ser Leu Ser Ala Leu 1 5 10 <210> 57 <211> 120 <212> PRT <213> Homo sapiens <400> 57 Gln Val Gln Leu Gln Glu Ser Gly Ser Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Gly Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Val Gln Leu Trp Gln Arg Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 58 <211> 112 <212> PRT <213> Homo sapiens <400> 58 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Asn Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 59 <211> 10 <212> PRT <213> Homo sapiens <400> 59 Gly Gly Ser Ile Ser Ser Ser Gly Tyr Tyr 1 5 10 <210> 60 <211> 7 <212> PRT <213> Homo sapiens <400> 60 Ile Tyr His Ser Gly Ser Thr 1 5 <210> 61 <211> 12 <212> PRT <213> Homo sapiens <400> 61 Ala Arg Val Gln Leu Trp Gln Arg Gly Phe Asp Tyr 1 5 10 <210> 62 <211> 11 <212> PRT <213> Homo sapiens <400> 62 Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr 1 5 10 <210> 63 <211> 3 <212> PRT <213> Homo sapiens <400> 63 Leu Gly Ser One <210> 64 <211> 9 <212> PRT <213> Homo sapiens <400> 64 Met Gln Ala Leu Gln Ala Pro Leu Thr 1 5 <210> 65 <211> 115 <212> PRT <213> Homo sapiens <400> 65 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Thr Trp Ser Asp Tyr Arg Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 66 <211> 215 <212> PRT <213> Homo sapiens <400> 66 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Arg 20 25 30 Arg Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Arg Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 67 <211> 8 <212> PRT <213> Homo sapiens <400> 67 Gly Gly Ser Phe Ser Gly Tyr Tyr 1 5 <210> 68 <211> 7 <212> PRT <213> Homo sapiens <400> 68 Ile Asn His Ser Gly Ser Thr 1 5 <210> 69 <211> 9 <212> PRT <213> Homo sapiens <400> 69 Val Thr Trp Ser Asp Tyr Arg Asp Tyr 1 5 <210> 70 <211> 7 <212> PRT <213> Homo sapiens <400> 70 Gln Ser Val Thr Ser Arg Arg 1 5 <210> 71 <211> 3 <212> PRT <213> Homo sapiens <400> 71 Gly Ala Ser One <210> 72 <211> 9 <212> PRT <213> Homo sapiens <400> 72 Gln Gln Tyr Gly Ser Ser Pro Leu Thr 1 5 <210> 73 <211> 126 <212> PRT <213> Homo sapiens <400> 73 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Gly Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Thr Thr Tyr Ser Tyr Gly Pro Gly Phe Asp Pro Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 <210> 74 <211> 216 <212> PRT <213> Homo sapiens <400> 74 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Tyr 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Val Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Thr Gln Arg Pro Ser Glu Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr Ser Val Thr Leu Asp Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Gly Asp Tyr Tyr Cys Ala Thr Trp Asp Ser Asn Leu 85 90 95 Lys Ser Val Val Phe Gly Gly Gly Thr Lys Val Ala Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 75 <211> 7 <212> PRT <213> Homo sapiens <400> 75 Ile Tyr Tyr Ser Gly Gly Thr 1 5 <210> 76 <211> 13 <212> PRT <213> Homo sapiens <400> 76 Ala Arg Thr Thr Tyr Ser Tyr Gly Pro Gly Phe Asp Pro 1 5 10 <210> 77 <211> 8 <212> PRT <213> Homo sapiens <400> 77 Ser Ser Asn Ile Gly Thr Tyr Tyr 1 5 <210> 78 <211> 3 <212> PRT <213> Homo sapiens <400> 78 Asp Asn Thr One <210> 79 <211> 11 <212> PRT <213> Homo sapiens <400> 79 Ala Thr Trp Asp Ser Asn Leu Lys Ser Val Val 1 5 10 <210> 80 <211> 126 <212> PRT <213> Homo sapiens <400> 80 Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Val Met Tyr Tyr Tyr Gly Ser Gly Ser Pro Tyr Trp Tyr 100 105 110 Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 81 <211> 181 <212> PRT <213> Homo sapiens <400> 81 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Lys Asn 20 25 30 Phe Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Phe Leu 35 40 45 Ile Tyr Glu Asn Ser Lys Arg Pro Ser Gly Ile Pro Asp Arg Ile Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr 180 <210> 82 <211> 10 <212> PRT <213> Homo sapiens <400> 82 Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr 1 5 10 <210> 83 <211> 7 <212> PRT <213> Homo sapiens <400> 83 Ile Tyr His Ser Gly Ser Thr 1 5 <210> 84 <211> 18 <212> PRT <213> Homo sapiens <400> 84 Ala Arg Val Met Tyr Tyr Tyr Gly Ser Gly Ser Pro Tyr Trp Tyr Phe 1 5 10 15 Asp Leu <210> 85 <211> 8 <212> PRT <213> Homo sapiens <400> 85 Ser Ser Asn Ile Gly Lys Asn Phe 1 5 <210> 86 <211> 3 <212> PRT <213> Homo sapiens <400> 86 Glu Asn Ser One <210> 87 <211> 122 <212> PRT <213> Homo sapiens <400> 87 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Leu Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Val Pro Ala Ile Arg Ser Ser Ala Ile Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 88 <211> 217 <212> PRT <213> Homo sapiens <400> 88 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Phe Gly Ser Arg Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Gln Leu Leu 35 40 45 Ile Phe Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Gly Ile Thr Gly Val Gln 65 70 75 80 Thr Gly Asp Glu Ala Glu Tyr Tyr Cys Gly Ala Trp Asp Ser Ser Leu 85 90 95 Ser Ala Gly Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 115 120 125 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 130 135 140 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 145 150 155 160 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 165 170 175 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 180 185 190 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 195 200 205 Lys Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 89 <211> 14 <212> PRT <213> Homo sapiens <400> 89 Ala Arg Val Pro Ala Ile Arg Ser Ser Ala Ile Phe Asp Tyr 1 5 10 <210> 90 <211> 8 <212> PRT <213> Homo sapiens <400> 90 Arg Ser Asn Ile Gly Asn Asn Tyr 1 5 <210> 91 <211> 12 <212> PRT <213> Homo sapiens <400> 91 Gly Ala Trp Asp Ser Ser Leu Ser Ala Gly Ala Val 1 5 10 <210> 92 <211> 121 <212> PRT <213> Homo sapiens <400> 92 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Ala Pro Tyr Ser Ser Ser Pro Ser Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 93 <211> 216 <212> PRT <213> Homo sapiens <400> 93 Leu Pro Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Arg Asn 20 25 30 Thr Gly Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Ala Glu Cys Ser 210 215 <210> 94 <211> 9 <212> PRT <213> Homo sapiens <400> 94 Gly Tyr Ser Ile Ser Ser Gly Tyr Tyr 1 5 <210> 95 <211> 7 <212> PRT <213> Homo sapiens <400> 95 Ile Tyr His Ser Gly Ser Thr 1 5 <210> 96 <211> 14 <212> PRT <213> Homo sapiens <400> 96 Ala Arg Val Ala Pro Tyr Ser Ser Ser Pro Ser Phe Asp Tyr 1 5 10 <210> 97 <211> 8 <212> PRT <213> Homo sapiens <400> 97 Ser Ser Asn Ile Gly Arg Asn Thr 1 5 <210> 98 <211> 3 <212> PRT <213> Homo sapiens <400> 98 Ser Asn Asn One <210> 99 <211> 11 <212> PRT <213> Homo sapiens <400> 99 Ala Ala Trp Asp Asp Ser Leu Asn Gly Val Val 1 5 10 <210> 100 <211> 118 <212> PRT <213> Homo sapiens <400> 100 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Phe Ser Arg Gly Gly Pro Phe Leu Trp Gly Arg Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 101 <211> 216 <212> PRT <213> Homo sapiens <400> 101 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Tyr 20 25 30 Ser Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala Tyr Val Phe Gly Ser Gly Thr Lys Val Thr Val Leu Gly Gln 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser 210 215 <210> 102 <211> 10 <212> PRT <213> Homo sapiens <400> 102 Ala Arg Phe Ser Arg Gly Gly Pro Phe Leu 1 5 10 <210> 103 <211> 8 <212> PRT <213> Homo sapiens <400> 103 Ser Ser Asn Ile Gly Ser Tyr Ser 1 5

Claims (31)

SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and human heavy chain variable framework, and SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and As an anti-marinobufagenin antibody comprising a human light chain variable framework (human light chain variable framework),
The anti-marinobufagenin antibody is selected from the group consisting of H1L1, H1L2, H2L1, H2L2, H3L1, and H3L2, anti-marinobufagenin antibody.
The method of claim 1,
The anti-marinobupagenin antibody is H1L1, the anti-marinobupagenin antibody.
The method of claim 1,
The anti-marinobupagenin antibody is H2L1, the anti-marinobupagenin antibody.
The method of claim 1,
The anti-marinobupagenin antibody is H3L1, anti-marinobupagenin antibody.
The method of claim 1,
The anti-marinobupagenin antibody is H1L2, anti-marinobupagenin antibody.
The method of claim 1,
The anti-marinobupagenin antibody is H2L2, anti-marinobupagenin antibody.
The method of claim 1,
The anti-marinobufagenin antibody is H3L2, anti-marinobufagenin antibody.
As a humanized anti-marinobufagenin antibody, the antibody is an antibody comprising a heavy chain variable region of SEQ ID NO: 1 and a light chain variable region of SEQ ID NO: 2 for binding to marinobufagenin Humanized anti-marinobufagenin antibody, which is a competitive inhibitor.
As a humanized anti-marinobufagenin antibody, the antibody is a heavy chain variable region selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 and SEQ ID NO : 12 and SEQ ID NO: The humanized anti-marinobupagenin antibody that competes for binding to the antibody comprising a light chain variable region selected from the group consisting of 13 and marinobufagenin.
The method of claim 9,
The antibody is a humanized anti-marinobufagenin antibody comprising the heavy chain variable region of SEQ ID NO: 11 and the light chain variable region of SEQ ID NO: 13.
The method of claim 1,
Wherein the antibody is an antibody fragment, anti-marinobufagenin antibody.
The method of claim 11,
Wherein the antibody fragment is a single chain antibody (single chain antibody), anti-marinobufagenin antibody.
The method of claim 11,
The antibody fragment is a Fab fragment, anti-marinobufagenin antibody.
The method of claim 11,
Wherein the antibody fragment is a recombinant fragment (recombinant fragment), anti-marinobufagenin antibody.
The method according to any one of claims 11 to 14,
Wherein the antibody fragment comprises a protracting moiety, anti-marinobufagenin antibody.
The method of claim 15,
The protruding moiety is polyethylene glycol, anti-marinobufagenin antibody.
The method according to any one of claims 1, 8, and 9,
The antibody is an IgG, anti-marinobufagenin antibody.
The method of claim 17,
Wherein the antibody is IgG1, anti-marinobufagenin antibody.
The method of claim 17,
Wherein the antibody is IgG2, anti-marinobufagenin antibody.
The method of claim 17,
Wherein the antibody is IgG4, anti-marinobufagenin antibody.
The method of claim 1,
The anti-marinobupagenin antibody is bound to the epitope of marinobufagenin bound by the 3E9 antibody, anti-marinobupagenin antibody.
As a method of treating pre-eclampsia, comprising the steps of:
Providing a mammal, wherein the mammal has pre-eclampsia and has an increased level of marinobufagenin; And
The step of administering an anti-marinobufagenin antibody to the mammal, wherein the anti-marinobupagenin antibody binds to marinobupagenin to treat pre-eclampsia.
The method of claim 22,
The mammal is a mouse, rat, or human being, the method of treating preeclampsia.
The method of claim 22 or 23,
The method of treating preeclampsia, wherein the anti-marinobufagenin antibody is a human antibody or a humanized antibody.
The method of claim 24,
The human antibodies are 201/202, 203/204, 205/207, 206/208, 236/237, 238/239, 240/241, R4CE-1E, R4CE-7G, R4CE-10H, 1B-6C, 1B- 6E, 2A-5A, and 2A-F8 will be selected from the group consisting of, a method of treating pre-eclampsia.
The method of claim 24,
The humanized antibody is selected from the group consisting of H1L1, H2L1, H3L1, H1L2, H2L2, and H3L2.
As a method of treating fibrosis, comprising the steps of:
Providing a mammal, wherein the mammal has fibrosis and has an increased level of marinobufagenin; And
The step of administering an anti-marinobufagenin antibody to the mammal, wherein the anti-marinobufagenin antibody binds to marinobufagenin to treat fibrosis.
The method of claim 27,
The method of treating fibrosis, wherein the mammal is a mouse, rat, or human.
The method of claim 27 or 28,
The method of treating fibrosis, wherein the anti-marinobufagenin antibody is a human antibody or a humanized antibody.
The method of claim 29,
The human antibodies are 201/202, 203/204, 205/207, 206/208, 236/237, 238/239, 240/241, R4CE-1E, R4CE-7G, R4CE-10H, 1B-6C, 1B- 6E, 2A-5A, and 2A-F8 will be selected from the group consisting of, a method of treating fibrosis.
The method of claim 29,
The humanized antibody is selected from the group consisting of H1L1, H2L1, H3L1, H1L2, H2L2, and H3L2.

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